WO2008101186A1 - Inhibiteurs de hdac8 - Google Patents

Inhibiteurs de hdac8 Download PDF

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Publication number
WO2008101186A1
WO2008101186A1 PCT/US2008/054126 US2008054126W WO2008101186A1 WO 2008101186 A1 WO2008101186 A1 WO 2008101186A1 US 2008054126 W US2008054126 W US 2008054126W WO 2008101186 A1 WO2008101186 A1 WO 2008101186A1
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Prior art keywords
optionally substituted
hdac8
compound
polypeptide
phenyl
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PCT/US2008/054126
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English (en)
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Eric Verdin
Scott Ulrich
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The J. David Gladstone Institutes
Ithaca College
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Publication of WO2008101186A1 publication Critical patent/WO2008101186A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C259/00—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups
    • C07C259/04—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids
    • C07C259/06—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids having carbon atoms of hydroxamic groups bound to hydrogen atoms or to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/38—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals

Definitions

  • Histone lysine acetylation patterns affect diverse cellular processes including differentiation, cellular response to stimuli and tumorigenesis.
  • HATs histone acetyl transferase
  • HDACs histone deacetylases
  • HDAC HDAC
  • Class III HDACs Sirtuins 1-7) are NAD + -dependent and unrelated in sequence to classes I and II (Holbert and Marmorstein 2005, Curr Opin Struct Biol 15:673-680). HDACs catalyze the removal of acetyl groups from lysine residues near the N-termini of hi stones.
  • Zinc-dependent HDACs have received much attention as anticancer drug targets. Inhibitors of these enzymes show a remarkable ability to induce terminal differentiation of transformed cells, presumably by altering patterns of gene expression through influencing the acetylation state of selected histone lysine residues (Marks et al, 2004, Adv Cancer Res 91 : 137-168). HDAC inhibitors are also exceedingly useful as tools to study the biology of histone deacetylases. Indeed, determining whether a cellular process involves HDACs is readily ascertained by using potent, cell-permeable molecules available.
  • HDAC inhibitors such as trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bishydroxamide (CBHA) act as competitive inhibitors; they closely mimic the aliphatic acetyl-lysine substrate and deliver a hydroxamic acid or other zinc-binding group to the catalytic zinc at the bottom of a narrow active site pocket as seen in the co-crystal structures of inhibited HDLP (HDAC -like protein; Finnin et al, 1999, Nature 401 : 188-193), HDAH (HDAC-like aminohydrolase; Nielsen et al, 2005, J Mo/5/o/ 354: 107-120), and human HDAC 8 (Somoza et ⁇ /., 2004, Structure 12: 1325-1334; Vannini et al, 2004, Proc Natl Acad Sci USA 101(42): 15064-9).
  • TSAHA suberoylanilide hydrox
  • TSA, SAHA, CBHA, 5-(4-methyl-benzoylamino)-biphenyl-3,4'-dicarboxylic acid 3-dimethylamide 4'-hydroxyamide (CRA-A), and 4-dimethylamino-N-(6- hydroxycarbamoyethyl)benzamide-N-hydroxy-7-(4- dimethylaminobenzoyl)aminoheptanamide (MS-344) are nonselective or poorly selective, i.e., they active against class I and class II HDACs and induce multiple effects within cells, including cell differentiation, induction of cell cycle arrest, and suppression of tumor growth (Jung et ⁇ /., 1999, J Med Chem 42:4669-4679; Almenara et ⁇ /., 2002, Leukemia 16: 1331- 1343; Richon et ⁇ /., 1998, Proc Natl Acad Sci USA 9:3003-3007; Yoshida et ⁇ /.
  • a set of inhibitors specific for each individual HDAC family member would enable the cellular role of each to be determined as well as help uncover the mechanism of the anticancer properties of HDAC inhibitors. Further, identifying selective HDAC inhibitors may open the way for developing pharmaceutical compositions with enhanced efficacy and/or tolerability.
  • the invention described herein provides for the rational design of HDAC inhibitors based on a new scaffold.
  • the newly designed HDAC inhibitors show remarkable selectivity towards human HDAC8 by targeting an active site pocket unique to this HDAC family member.
  • this invention discloses a new HDAC inhibitor scaffold designed to exploit a unique sub-pocket of the HDAC8 active site. These compounds are based on inspection of HDAC8 crystal structures bound to various inhibitors, which showed that the HDAC8 active site is surprisingly malleable and can accommodate inhibitor structures that are distinct from the canonical "zinc-binding group-linker-cap group" structures of SAHA, TSA and similar HDAC inhibitors. Some of the new inhibitors based on this new scaffold are >100 fold selective for HDAC8 over other class I and class II HDACs with IC50 values ⁇ 1 ⁇ M against HDAC8.
  • the present invention provides a new type of "linkerless" HDAC8 inhibitors and methods of treating a pathological condition using the same.
  • this invention provides a compound having the formula
  • this invention provides a compound having the formula in which Z is -NH, O or S,
  • R 1 represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate,
  • R 2 is an optionally substituted phenyl or naphthyl group in which the substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxy
  • this invention provides a compound having the formula
  • R 3 and R 4 are independently hydrogen, lower haloalkyl, lower alkyl, — CO 2 H, NO 2 , lower alkyl carboxylate, lower alkoxy, or — CN.
  • the invention provides a compound of the formula
  • the invention provides a compound of the formula
  • the invention provides a compound of the formula
  • R 3 and R 4 are independently hydrogen, lower haloalkyl, lower alkyl, — CO 2 H, — NO 2 , lower alkyl carboxylate, lower alkoxy, or — CN.
  • the present invention provides a compound having the formula
  • the present invention provides a compound having the formula
  • the present invention also provides pharmaceutical compositions comprising a compound of the invention and one or more pharmaceutically acceptable excipients.
  • the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula
  • the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula
  • R 1 represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate,
  • the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula
  • R 3 and R 4 are independently hydrogen, lower haloalkyl, lower alkyl, — CO 2 H, — NO 2 , lower alkyl carboxylate, lower alkoxy, or — CN and (ii) a pharmaceutically acceptable excipient or carrier.
  • the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula
  • the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula
  • the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula
  • the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula
  • the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula
  • X is oxygen or sulphur and (ii) a pharmaceutically acceptable excipient or carrier.
  • the present invention also provides a method for selectively inhibiting an activity of an HDAC8 polypeptide.
  • the method comprises the step of contacting an HDAC8 polypeptide with an effective inhibiting amount of a compound having the formula:
  • W represents a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocy
  • X represents a bond, an optionally substituted C 1 -C 3 alkylene group, an optionally substituted C 2 -C 3 alkenylene group or -C ⁇ C-, in which optional substituents are selected from one or more of halogen, hydroxy, lower alkoxy, lower alkylthio, lower haloalkoxy, and lower haloalkylthio; and
  • Y represents a zinc-binding moiety
  • R 1 represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are as defined for moiety W;
  • R 2 is an optionally substituted phenyl or naphthyl group in which substituents are as defined for moiety W.
  • the zinc binding moiety may be a hydroxamic acid group or a hydroxamic acid derivative.
  • the compound is selected from the group consisting of compounds of the formula
  • R 1 represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate,
  • R 2 is an optionally substituted phenyl or naphthyl group in which the substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxy
  • R 3 and R 4 are independently hydrogen, lower haloalkyl, lower alkyl, — CO 2 H, — NO 2 , lower alkyl carboxylate, lower alkoxy, or — CN,
  • R 3 and R 4 are independently hydrogen, lower haloalkyl, lower alkyl, — CO 2 H, — NO 2 , lower alkyl carboxylate, lower alkoxy, or — CN,
  • a preferred activity of the HDAC8 polypeptide is a histone deacetylase activity or a tubulin deacetylase activity.
  • a preferred compound of the present invention inhibits the HDAC8 polypeptide with an efficiency of greater than 50 fold over HDACl or HDAC6
  • a preferred compound of the present invention inhibits the HDAC8 polypeptide with an IC50 of less than 1 ⁇ M.
  • the method for selectively inhibiting an activity of an HD AC8 polypeptide may be practiced in vitro or in vivo.
  • the compound is provided as a prodrug.
  • the present invention provides a method for regulating smooth muscle cell contraction in an animal.
  • this method comprises the step of administering to an animal an effective amount of a compound or a pharmaceutically acceptable salt thereof, the compound having the formula
  • W represents a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocy
  • X represents a bond, an optionally substituted C 1 -C 3 alkylene group, an optionally substituted C 2 -C 3 alkenylene group or -C ⁇ C-, in which optional substituents are selected from one or more of halogen, hydroxy, lower alkoxy, lower alkylthio, lower haloalkoxy, and lower haloalkylthio; and
  • Y represents a zinc-binding moiety
  • R 1 represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are as defined for moiety W;
  • R 2 is an optionally substituted phenyl or naphthyl group in which substituents are as defined for moiety W.
  • the present invention provides a method for treating a pathological condition characterized by an aberrant genetic repression of gene expression.
  • this method comprises the step of administering to an animal an effective amount of a compound or a pharmaceutically acceptable salt thereof, the compound having the formula
  • W represents a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocy
  • X represents a bond, an optionally substituted C 1 -C 3 alkylene group, an optionally substituted C 2 -C 3 alkenylene group or -C ⁇ C-, in which optional substituents are selected from one or more of halogen, hydroxy, lower alkoxy, lower alkylthio, lower haloalkoxy, and lower haloalkylthio; and
  • Y represents a zinc-binding moiety
  • R 1 represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are as defined for moiety W;
  • R 2 is an optionally substituted phenyl or naphthyl group in which the substituents are as defined for moiety W.
  • the animal is a human.
  • Preferred pathological conditions are cancer and acute myeloid leukemia.
  • Figure 1 depicts common structural features of HDAC inhibitors SAHA and TSA, such as cap group, linker and zinc-binding group.
  • FIG. 2 depicts an HDAC8:TSA co-crystal structure showing the conserved hydrophobic amino acid residues (F 152, F208, and M274) which form the narrow active site channel, at the bottom of which is the catalytic zinc ion. Also shown is a TSA molecule entering the narrow active site channel.
  • Figure 3 A depicts the structure of HDAC8 bound to SAHA, an alkyl-linker HDAC inhibitor. Amino acid residues M274 and F 152 (indicated by arrows) pack against each other to form the wall of the active site pocket.
  • Figure 3B depicts the structure of HDAC8 bound to CRA-A, an aryl-linker HDAC inhibitor. In this case amino acid residue F 152 rotates away from amino acid residue M274, exposing a large sub-pocket.
  • Figure 4 depicts the structures of Compounds 1 to 6, linkerless, sterically demanding hydroxamates designed to bind the sub-pocket of the F1DAC8 active site.
  • Figure 5 depicts a scheme for the synthesis of hydroxamates shown in Figure 4. Details are described in Examples 2 and 3.
  • Figure 6 depicts selective inhibition of Compounds 2, 5, and 6 for HDAC8.
  • Figure 7 depicts that cellular proteins in human cells are acetylated in response to treatment with TSA, Compound 2 or Compound 5.
  • A. and B. Anti-acetyl lysine Western blot of HeLa and HEK293 cell lysates pretreated with TSA (a relatively non-specific HDAC inhibitor), no inhibitor ("0") and increasing concentrations of Compound 2 (panel A) and
  • Figure 8 depicts an HDAC8: CRA-A co-crystal structure showing the right-angle orientation of the aryl linker relative to the induced sub-pocket.
  • the aryl group of CRA-A bearing the hydroxamic acid does not bind the sub-pocket but rather is positioned well above it, as can be seen in Fig. 3B.
  • Figures 9A and 9B show reaction schemes for the making of a phenyl -substituted pyrrol (e.g., Compound 7). See also Example 4 for details.
  • Figure 1OA shows a reaction scheme for making naphthyl-substituted pyrroles, furans and thiophenes (e.g., Compounds 12-15, wherein S is changed to O or N) using a naphthyl boronic acid.
  • Figure 1OB shows a reaction scheme for making compounds 10 and 11 as described in Example 5 DETAILED DESCRIPTION OF THE INVENTION
  • acetylation status refers to the presence or absence of an acetyl group on a polypeptide, preferably a histone polypeptide.
  • activity of HDAC8 refers to (i) binding of an HDAC8 polypeptide to a polypeptide or peptide, preferably a histone or tubulin polypeptide or a cellular polypeptide, (ii) interaction of an HDAC8 polypeptide with a polypeptide or peptide, preferably a histone or tubulin polypeptide or a cellular polypeptide, (iii) assembly of an HDAC8 polypeptide into a multiprotein complex, preferably a multiprotein complex comprising an inversion(l ⁇ ) protein product or a cellular polypeptide, (iv) localization of an HDAC8 polypeptide in the cytoplasm of a eukaryotic cell, or (v) enzymatic deacetylating of an acetylated polypeptide or peptide, preferably a histone or tubulin polypeptide.
  • active site of HDAC8 or grammatical equivalents thereof refer to a region of an HDAC8 polypeptide surface within 50 angstroms of the zinc ion.
  • an "agent” or “candidate agent” can be any chemical compound, for example, a macromolecule or a small molecule.
  • the candidate agent can have a formula weight of less than about 10,000 grams per mole, less than 5,000 grams per mole, less than 1,000 grams per mole, or less than about 500 grams per mole.
  • the candidate agent can be naturally occurring (e.g., a herb or a nature product), synthetic, or both.
  • macromolecules are proteins, protein complexes, and glycoproteins, nucleic acids, e.g., DNA, RNA and PNA (peptide nucleic acid).
  • small molecules are peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds e.g., heteroorganic or organometallic compounds.
  • a candidate agent can be the only substance assayed by the method described herein. Alternatively, a collection of candidate agents can be assayed either consecutively or concurrently by the methods described herein.
  • Candidate agents encompass numerous chemical classes, typically synthetic, semisynthetic, or naturally-occurring inorganic or organic molecules.
  • Candidate agents may be small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
  • Candidate agents may comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, and may contain at least two of the functional chemical groups.
  • the candidate agents may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • alkyl refers to a straight or branched chain saturated hydrocarbon moiety, and can include di- and multivalent groups, having the number of carbon atoms designated (i.e. C 1 -C 1 O means one to ten carbons).
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, and the various pentyl, hexyl, heptyl, octyl, etc. groups.
  • lower alkyl refers to alkyl groups having from 1 to 4 carbon atoms.
  • haloalkyl refers to alkyl groups substituted by one or more halogen atoms, which may be the same or different.
  • alkoxy and alkylthio refer to oxygen and sulfur atoms, respectively, substituted by alkyl groups of the indicated number of carbon atoms; “haloalkoxy” and “haloalkylthio” refer to such groups substituted by one or more halogens, which may be the same or different.
  • alklysulfonyl and haloalkylsulfonyl refer to sulfonyl (-SO 2 -) moieties substituted by an alkyl or haloalkyl group, respectively.
  • alkylene refers to a divalent group derived from an alkyl group and includes, e.g., methylene, -CH 2 -, ethylene, -CH 2 CH 2 -, propylene, - CH 2 CH 2 CH 2 - and the like.
  • alkenyl refers to a straight or branched chain unsaturated hydrocarbyl moiety having one or more double bonds.
  • alkenyl groups include vinyl, allyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl and 3 -(1,4- pentadienyl).
  • lower alkenyl refers to alkenyl groups having from 2 to 4 carbon atoms.
  • alkylene by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified by -CH 2 CH 2 CH 2 CH 2 -, and further includes those groups described below as “heteroalkylene.”
  • an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • alkynyl refers to an unsaturated alkyl group one having 2-4 carbon atoms and a triple bond.
  • alkynyl groups include ethynyl (acetylenyl), 1-propynyl, 1- and 2-butynyl.
  • antagonist means a chemical substance that diminishes, abolishes or interferes with the physiological action of a polypeptide.
  • the antagonist may be, for example, a chemical antagonist, a pharmacokinetic antagonist, a non-competitive antagonist, or a physiological antagonist, such as a biomolecule, e.g., a polypeptide.
  • a preferred antagonist diminishes, abolishes or interferes with the physiological action of an HDAC8 polypeptide.
  • an antagonist may act at the level of the interaction between a first polypeptide, e.g., an HDAC8 polypeptide and a second polypeptide, for example, a binding partner, such as a histone polypeptide, an inversion(l ⁇ ) protein product or a cellular polypeptide.
  • the antagonist may by competitively or non-competitively (e.g., allosterically) inhibit binding of the first polypeptide to the second polypeptide.
  • a "pharmacokinetic antagonist" effectively reduces the concentration of the active drug at its site of action, e.g., by increasing the rate of metabolic degradation of the first polypeptide.
  • a “competitive antagonist” is a molecule which binds directly to the first polypeptide in a manner that sterically interferes with the interaction of the first polypeptide with the second polypeptide.
  • Non-competitive antagonism describes a situation where the antagonist does not compete directly with the binding, but instead blocks a point in the signal transduction pathway subsequent to the binding of the first polypeptide to the second polypeptide.
  • an antagonist can also be a substance that diminishes or abolishes expression of a first polypeptide.
  • an HDAC8 antagonist can be, for example, a substance that diminishes or abolishes: (i) the expression of the gene encoding HDAC8, (ii) the translation of HDCA8 RNA, (iii) the post-translational modification of HDAC8, such as phosphorylation, or (iv) the interaction of an HDAC8 polypeptide with other polypeptides in the formation of a multi-protein complex.
  • aryl means, unless otherwise stated, a polyunsaturated, typically aromatic, hydrocarbon substituent which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently.
  • heteroaryl refers to aryl groups (or rings) that contain from zero to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized.
  • a heteroaryl group can be attached to the remainder of the molecule through a heteroatom.
  • Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2- imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5- benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinoly
  • aryl when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above.
  • arylalkyl is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l- naphthyloxy)propyl, and the like).
  • alkyl group e.g., benzyl, phenethyl, pyridylmethyl and the like
  • an oxygen atom e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l- naphthyloxy
  • R', R" and R'" each independently refer to hydrogen, unsubstituted (Ci-C8)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(Ci-C 4 )alkyl groups.
  • R' and R" When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.
  • -NR'R is meant to include 1-pyrrolidinyl and 4-morpholinyl.
  • alkyl is meant to include groups such as haloalkyl (e.g., -CF 3 and - CH 2 CF 3 ) and acyl (e.g., -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 , and the like).
  • Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(0)-(CH 2 ) q -U-, wherein T and U are independently -NH-, -0-, -CH 2 - or a single bond, and q is an integer of from O to 2.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are independently -CH 2 -, -0-, -NH-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR'- or a single bond, and r is an integer of from 1 to 3.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH 2 ) S - X-(CH 2 ) r , where s and t are independently integers of from O to 3, and X is -0-, -NR'-, -S-, - S(O)-, -S(O) 2 -, or -S(O) 2 NR'-.
  • the substituent R' in -NR'- and -S(O) 2 NR'- is selected from hydrogen or unsubstituted (Ci-C 6 )alkyl.
  • association with HDAC8 means contact, interact, or bind to HDAC8.
  • biologically active when referring to an agent or compound is art-recognized and refers to a form of an agent or compound that allows for it, or a portion of the amount of agent or compound administered, to be absorbed by, incorporated to, or otherwise physiologically available to a subject or patient to whom it is administered.
  • biological sample means a sample of biological tissue or fluid that contains nucleic acids or polypeptides. Such samples are typically from humans, but include tissues isolated from non-human primates, or rodents, e.g., mice, and rats. Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histological purposes, cerebral spinal fluid, blood, plasma, serum, sputum, stool, tears, mucus, hair, skin, etc. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues. A “biological sample” also refers to a cell or population of cells or a quantity of tissue or fluid from an animal.
  • biological sample can also refer to cells or tissue analyzed in vivo, i.e., without removal from the animal.
  • a biological sample will contain cells from the animal, but the term can also refer to noncellular biological material, such as noncellular fractions of, blood, serum, saliva, cerebral spinal fluid or urine, that can be used to measure expression level of a polynucleotide or polypeptide.
  • noncellular biological material such as noncellular fractions of, blood, serum, saliva, cerebral spinal fluid or urine, that can be used to measure expression level of a polynucleotide or polypeptide.
  • Numerous types of biological samples can be used in the present invention, including, but not limited to, a tissue biopsy or a blood sample.
  • tissue biopsy refers to an amount of tissue removed from an animal, preferably a human, for diagnostic analysis.
  • tissue biopsy can refer to any type of biopsy, such as needle biopsy, fine needle biopsy, surgical biopsy, etc.
  • Providing a biological sample means to obtain a biological sample for use in methods described in this invention. Most often, this will be done by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells ⁇ e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo. Archival tissues, having treatment or outcome history, will be particularly useful.
  • “Cancer,” “cancer cells,” “transformed” cells or “transformation” in tissue culture refers to spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material.
  • transformation can arise from infection with a transforming virus and incorporation of new genomic DNA, or uptake of exogenous DNA, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation is associated with phenotypic changes, such as immortalization of cells, aberrant growth control, nonmorphological changes, and/or malignancy ⁇ see, Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed. 1994)).
  • change in cell growth refers to any change in cell growth and proliferation characteristics in vitro or in vivo, such as formation of foci, anchorage independence, semi-solid or soft agar growth, changes in contact inhibition and density limitation of growth, loss of growth factor or serum requirements, changes in cell morphology, gaining or losing immortalization, gaining or losing tumor specific markers, ability to form or suppress tumors when injected into suitable animal hosts, and/or immortalization of the cell. See, e.g., Freshney, Culture of Animal Cells a Manual of Basic Technique pp. 231-241 (3 rd ed. 1994).
  • a "combinatorial chemical library” refers to a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • cycloalkyl refers to a saturated cyclic hydrocarbon having 3 to 8 carbon atoms, and 1 to 3 rings that can be fused or linked covalently.
  • Cycloalkyl groups useful in the present invention include, but are not limited to, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
  • Bicycloalkyl groups useful in the present invention include, but are not limited to, [3.3.0]bicyclooctanyl, [2.2.2]bicyclooctanyl, [4.3.0]bicyclononane, [4.4.0]bicyclodecane (decalin), spiro[3.4]octanyl, spiro[2.5]octanyl, and so forth.
  • cycloalkenyl refers to an unsaturated cyclic hydrocarbon having 3 to 15 carbons, and 1 to 3 rings that can be fused or linked covalently.
  • Cycloalkenyl groups useful in the present invention include, but are not limited to, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl. Bicycloalkenyl groups are also useful in the present invention.
  • the term "decreased expression” refers to the level of a gene expression product that is lower and/or the activity of the gene expression product is lowered.
  • the decrease is at least 20%, more preferably, the decrease is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% and most preferably, the decrease is at least 100%, relative to a control.
  • determining are contemplated within the scope of the present invention and include, but are not limited to, detecting, measuring, assaying, testing or determining, the presence, absence, amount or concentration of a molecule, such as a an HDAC8, a label, or a compound of the invention and the like.
  • the term refers to both qualitative and quantitative determinations.
  • determining the effect or “determining the functional effect” means assaying for an agent or compound that increases or decreases a parameter that is indirectly or directly under the influence of the agent or compound, e.g., functional, enzymatic, physical and chemical effects.
  • Such effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein, measuring inducible markers or transcriptional activation of a gene, such as HDAC8; measuring binding activity, e.g., binding of an HDAC8 polypeptide to another polypeptide; assaying for deacetylation activity of HDAC8; determining the acetylation status of an HDAC8 substrate, such as a histone polypeptide, tubulin polypeptide or another cellular polypeptide; measuring cellular proliferation, measuring apoptosis, measuring subcellular localization of a polypeptide, such as HDAC8; or the like.
  • spectroscopic characteristics e.g., fluorescence, absorbance, refractive index
  • hydrodynamic e.g., shape
  • Determination of the functional effect of an agent or compound on a disease, disorder, cancer or other pathology can also be performed using assays known to those of skill in the art such as an in vitro assays, e.g., cellular proliferation; growth factor or serum dependence; mRNA and protein expression in cells, and other characteristics of cells.
  • assays known to those of skill in the art such as an in vitro assays, e.g., cellular proliferation; growth factor or serum dependence; mRNA and protein expression in cells, and other characteristics of cells.
  • the effects can be evaluated by many means known to those skilled in the art, e.g., microscopy for quantitative or qualitative measures of alterations in morphological features, measurement of changes in RNA or protein levels, measurement of RNA stability, identification of downstream or reporter gene expression (CAT, luciferase, ⁇ -gal, GFP and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays, apoptosis assays, measuring the production of acetyl-CoA and AMP, and the like.
  • “Functional effects” include in vitro, in vivo, and ex vivo activities.
  • disorders disorders
  • a specific disease is manifested by characteristic symptoms and signs, including biological, chemical and physical changes, and is often associated with a variety of other factors including, but not limited to, demographic, environmental, employment, genetic and medically historical factors. Certain characteristic signs, symptoms, and related factors can be quantitated through a variety of methods to yield important diagnostic information.
  • ⁇ ективное amount means a dosage sufficient to produce a desired result, to ameliorate, or in some manner, reduce a symptom or stop or reverse progression of a condition.
  • the desired result is an increase in cytoplasmic localization of an F1DAC8 polypeptide.
  • the desired result is an increase in nuclear localization of an HDAC8 polypeptide.
  • the desired result is an increase in the deacetylation activity of an HDAC8 polypeptide.
  • the desired result is an increase or decrease in the acetylation status of an HDAC8 substrate, such as a histone polypeptide or tubulin polypeptide.
  • Amelioration of a symptom of a particular condition by administration of a pharmaceutical composition described herein refers to any lessening, whether permanent or temporary, lasting or transit that can be associated with the administration of the pharmaceutical composition.
  • An "effective amount" can be administered in vivo and in vitro.
  • a "full length" polypeptide or nucleic acid refers to a polypeptide or polynucleotide sequence, or a variant thereof, that contains all of the elements normally contained in one or more naturally occurring, polynucleotide or polypeptide sequences.
  • the "full length” may be prior to, or after, various stages of post-translation processing or splicing, including alternative splicing and signal peptide cleavage.
  • the HDAC8 polypeptide is a "full length" HDAC8 polypeptide referring to a polypeptide that has at least the length of a naturally occurring HDAC8 polypeptide.
  • a "full-length" HDAC8 polypeptide or a fragment thereof can also include other sequences, e.g., a purification tag (such as FLAG or HA), or other attached compounds (such as an attached fluorophore, a label, or cofactor).
  • a purification tag such as FLAG or HA
  • other attached compounds such as an attached fluorophore, a label, or cofactor
  • halo or halogen
  • F fluorine
  • Cl chlorine
  • Br bromine
  • I iodine
  • haloalkyl monohaloalkyl
  • halo(Ci-C 4 )alkyl is mean to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
  • FIDAC histone deacetylase
  • F1DAC8 refers to nucleic acids, polypeptides and polymorphic variants, alleles, mutants, and interspecies homologues thereof and as further described herein, that: (1) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 75, 100, 150, 200, 250, 300, 350, or 377 amino acids, to an F1DAC8 sequence as deposited under GenBank Accession Nos., e.g., CAB90213, AAF73428, NP_060956, Q9BY41, AAF73076, AAH50433, AAH61257, AAL47569,
  • HDAC8 was cloned and characterized by several independent research groups (e.g., Van den Wyngaert et al, 2000, FEBS Lett 478(l-2):77-83; Buggy et al., 2000, Biochem J350 PtI : 199-205; Hu et al., 2000, J Biol Chem 275(20): 15254-64; incorporated herein by reference in their entireties).
  • HDAC8 polynucleotide or polypeptide sequence is typically from a human, but may be from other mammals, but not limited to, a non-human primate, a rodent, e.g., a rat, mouse, or hamster; a cow, a pig, a horse, a sheep, or other mammal.
  • rodent e.g., a rat, mouse, or hamster
  • cow, a pig, a horse, a sheep, or other mammal it is desirable to use an HDAC8 or HDAC8-like polynucleotide or polypeptide from yeast, Drosophila, trypanosome, chicken, C. elegans, or Xenopus.
  • An "HDAC8" polypeptide and polynucleotide includes both naturally occurring or recombinant forms.
  • an HDAC8 polypeptide and an HDAC8 subdomain polypeptide as described herein can comprise a sequence that corresponds to a human HDAC8 polypeptide sequence.
  • exemplary HDAC8 polypeptide sequences and are known in the art.
  • GenBank accession numbers for human HDAC8 polypeptides are AAF73428, NP 060956, Q9BY41, CAB90213, AAH50433, and AAF73076.
  • GenBank accession numbers for mouse HDAC8 polypeptides are, for example, AAH61257, Q8VH37, NP_081658, and AAL47569; for zebrafish HDAC8, NP-998596 and AAH55541.
  • Exemplary HDAC8 polynucleotide sequences are known in the art.
  • Exemplary, GenBank accession numbers for human HDAC8 nucleic acids are BC050433, NM_018486, AJ277724, AF245664, and AF230097.
  • GenBank accession numbers for mouse HDAC8 include BC061257, NT_039706, NM_027382, and AY066003; pig HDAC8, AY556472; and for zebrafish HDAC8, BC055541 and NM 213431.
  • HDAC8 fusion protein refers to a polypeptide that comprises (i) an amino acid sequence of an HDAC8, an HDAC8 fragment, an HDAC8 subdomain polypeptide, an HDAC8 related polypeptide or a fragment of an HDAC8 related polypeptide and (ii) an amino acid sequence of a heterologous polypeptide (i.e., a non-HDAC8, non- HDAC8 fragment or non-HDAC8 related polypeptide).
  • HDAC8 homolog refers to a polypeptide that comprises an amino acid sequence similar to that of HDAC8, but does not necessarily possess a similar or identical function as HD AC8.
  • HDAC8 isoform refers to a variant of HDAC8 that is encoded by the same gene, but differs in its pi or MW, or both. Such isoforms can differ in their amino acid composition (e.g., as a result of alternative splicing or limited proteolysis) and in addition, or in the alternative, may arise from differential post-translational modification (e.g., glycosylation, acetylation or phosphorylation).
  • HDAC8 ortholog refers to a non-human polypeptide that (i) comprises an amino acid sequence similar to that of human HDAC8 and (ii) possess a similar or identical function to that of human HDAC8.
  • an "HDAC8 substrate” refers to a polypeptide with which an HDAC8 polypeptide interacts or interacts and/or a polypeptide which is deacetylated by an HDAC polypeptide.
  • a preferred HDAC8 substrate is a histone polypeptide or tubulin polypeptide.
  • HDAC8 structure or “structure of HDAC8” refers to the crystal structure of HDAC8 as determined by Somoza et ah. (Somoza et ah., 2004, Structure 12: 1325- 1334; incorporated herewith by reference in its entirety) and/or Vannini et ah, (Vannini et ah., 2004, Proc Nath Acad Sci USA 101(42): 15064-15069; incorporated herewith by reference in its entirety).
  • heteroatom is meant to include oxygen (O), nitrogen (N), Boron (B), phosphorous (P) and sulfur (S).
  • heteroaryl refers to a polyunsaturated aromatic hydrocarbon substituent having 5-12 ring members, which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently, and which has at least one heteroatom in the ring, such as N, O, or S.
  • a heteroaryl group can be attached to the remainder of the molecule through a heteroatom.
  • heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, A- isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3- quinolyl, and 6-
  • Additional heteroaryl groups useful in the present invention include pyridyl N-oxide, tetrazolyl, benzofuranyl, benzothienyl, indazolyl, or any of the radicals substituted, especially mono- or di-substituted.
  • heteroatom or "ring heteroatom” is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
  • heterocycloalkyl refers to a saturated cyclic hydrocarbon having 3 to 15 ring members, and 1 to 3 rings that can be fused or linked covalently, and which has at least one heteroatom in the ring, such as N, O, or S. Additionally, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.
  • heterocycloalkyl examples include 1 -(1,2,5, 6-tetrahydropyridyl), 1-piperidinyl, 2- piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like.
  • a "host cell” is a naturally occurring cell or a transformed cell that contains an expression vector and supports the replication or expression of the expression vector.
  • Host cells may be cultured cells, explants, cells in vivo, and the like.
  • Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect cells, amphibian cells, or mammalian cells such as CHO, 293, 3T3, HeLa, and the like ⁇ see, e.g., the American Type Culture Collection catalog).
  • hydroxamic acid group or “hydroxamic acid” refer to an N- hydroxylated amide and its derivatives include any type of chemical functionality attached to the carbonyl of the hydroxamic acid.
  • the terms "individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines, simians, felines, canines, equines, bovines, mammalian farm animals, mammalian sport animals, and mammalian pets and humans. Preferred is a human. In certain embodiments, the terms also include Xenopus, zebrafish, trypanosome, C. elegans, Drosophila, and yeast.
  • inhibitor refers to an agent or compound that, e.g., represses or inactivates the expression of a polypeptide of the invention or binds to, decreases, closes, inactivates, impedes, or reduces activation, desensitizes or down regulates the activity of a polypeptide of the invention.
  • Inhibitors include nucleic acids such as siRNA, antisense RNA, and ribozymes that interfere with the expression of e.g., HDAC8 as well as naturally occurring and synthetic compounds and agents, small chemical molecules and the like.
  • Preferred HDAC8 inhibitors are the compounds described herein. Assays for inhibitors are described herein.
  • Samples or assays comprising e.g., an HDAC8 polypeptide that are treated with a potential inhibitor are compared to control samples without the inhibitor to examine the extent of the effect.
  • Control samples (untreated with candidate agents or compounds) are assigned a relative activity value of 100%.
  • Inhibition of the HDAC8 polypeptide is achieved when the level or activity value relative to a control is reduced by 10%, optionally 20%, optionally 30%, optionally 40%, optionally 50%, 60%, 70%, 80%, or 90-100%.
  • in vitro means outside the body of the organism from which a cell or cells is obtained or from which a cell line is isolated.
  • in vivo means within the body of the organism from which a cell or cells is obtained or from which a cell line is isolated.
  • isomers refers to compounds of the present invention that possess asymmetric carbon atoms (optical or chiral centers) or double bonds.
  • a “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include 1 H, 3 H, 125 1, 32 P, 13 C, and 14 C, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into an HDAC8 polypeptide, a substrate of an HDAC8 polypeptide, or a compound.
  • a preferred label is 14 C or 3 H, preferably in an acetyl group.
  • level of a mRNA in a biological sample refers to the amount of mRNA transcribed from a gene that is present in a cell or a biological sample.
  • the mRNA generally encodes a functional protein, although mutations may be present that alter or eliminate the function of the encoded protein.
  • a "level of mRNA” need not be quantified, but can simply be detected, e.g., a subjective, visual detection by a human, with or without comparison to a level from a control sample or a level expected of a control sample.
  • a preferred mRNA is an HDAC8 mRNA.
  • level of a polypeptide in a biological sample refers to the amount of polypeptide translated from a mRNA that is present in a cell or biological sample.
  • the polypeptide may or may not have protein activity.
  • a "level of a polypeptide” need not be quantified, but can simply be detected, e.g., a subjective, visual detection by a human, with or without comparison to a level from a control sample or a level expected of a control sample.
  • a preferred polypeptide is an HDAC8 polypeptide.
  • mammalian means or relates to the class mammalia including, but not limited to the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys).
  • carnivore e.g., dogs and cats
  • rodentia e.g., mice, guinea pigs, and rats
  • primates e.g., humans, chimpanzees, and monkeys.
  • multi-protein complex refers to the binding and non-covalent attachment of two or more polypeptides to each other.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature.
  • a naturally occurring nucleic acid molecule can encode a natural protein.
  • a "naturally-occurring" polypeptide refers to a polypeptide molecule having an amino acid sequence that occurs in nature.
  • pharmaceutically acceptable refers to compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction when administered to a subject, preferably a human subject.
  • pharmaceutically acceptable means approved by a regulatory agency of a Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • the term "pocket" within an HDAC8 polypeptide refers to any surface site of HDAC8 that is a binding site for a small molecule.
  • a “subpocket” within an HDAC8 polypeptide refers to the pocket adjacent to the active site between amino acid residues M274 and F 152.
  • polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer.
  • Preferred polypeptides are HDACs, in particular HDAC8.
  • a "purified” or “isolated” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. "Substantially free” means that the protein of interest in the preparation is at least 10% pure. In an embodiment, the preparation of the protein has less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of a contaminating component (e.g., a protein not of interest, chemical precursors, and so forth).
  • a contaminating component e.g., a protein not of interest, chemical precursors, and so forth.
  • culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • recombinant when used with reference to, e.g., a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.
  • nucleic acid By the term “recombinant nucleic acid” herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. In this manner, operably linkage of different sequences is achieved.
  • an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined are both considered recombinant for the purposes of this invention.
  • a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
  • a "recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.
  • salt refers to salt of an active compound or agent of the present invention, such as an HDAC8 inhibitor, which is prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
  • salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like ⁇ see, e.g., Berge et al., 1977, J Pharm Science 66: 1-19).
  • Certain specific agents of the present invention contain both basic and acidic functionalities that allow the agents to be converted into either base or acid addition salts.
  • the neutral forms of the compounds of the present invention may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound and agent for the purposes of the present invention.
  • the phrases "selective inhibition of HDAC8" or “selectively inhibiting HDAC8” or grammatical equivalents thereof refer to the inhibition of an HDAC8 polypeptide by a compound of the present invention which occurs at an IC 50 concentration of a the compound which is at least 2 fold less, at least 5 fold less, at least 10 fold less, at least 20 fold less, at least 50 fold less, or at least 100 fold less than the IC50 concentration of the same compound for HDACl or HDAC6.
  • subdomain refers to a fragment of that protein, such as a fragment of HDAC8 polypeptide, which participates in an interaction, e.g., an intramolecular or an inter-molecular interaction, e.g., a binding or catalytic interaction.
  • An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken).
  • An inter-molecular interaction can be between the protein and another protein, between the protein and another compound, or between a first molecule and a second molecule of the protein (e.g., a dimerization interaction).
  • Biologically active portions/functional domains of a protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the protein which include fewer amino acids than the full length, natural protein, and exhibit at least one activity of the natural protein.
  • Biological active portions/functional domains can be identified by a variety of techniques including truncation analysis, site-directed mutagenesis, and proteolysis. Mutants or proteolytic fragments can be assayed for activity by an appropriate biochemical or biological (e.g., genetic) assay.
  • a functional domain is independently folded.
  • biologically active fragments comprise a domain or motif with at least one activity of the protein, e.g., an HDAC8 core catalytic domain.
  • a biologically active portion/functional domain of a protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length.
  • Biologically active portions/functional domain of a protein such as an HDAC8 polypeptide (i) can bind to a histone polypeptide or inversion(l ⁇ ) protein product, (ii) have deacetylation activity, or (iii) assemble into a multiprotein complex, comprising e.g., a histone polypeptide or inversion(l ⁇ ) protein product, or (iv) are useful for treatment of a pathological condition as described herein.
  • a "related" polypeptide as used herein refers to a homolog, an isoform, an ortholog, a fusion protein or fragments of a polypeptide or any combination thereof.
  • solvate refers to compounds and agents of the present invention that are complexed to a solvent.
  • Solvents that can form solvates with the compounds and agents of the present invention include common organic solvents such as alcohols (methanol, ethanol, etc.), ethers, acetone, ethyl acetate, halogenated solvents (methylene chloride, chloroform, etc.), hexane and pentane. Additional solvents include water. When water is the complexing solvent, the complex is termed a "hydrate.”
  • subject or “patient” to be treated for a pathological condition, disorder, or disease by a subject method means either a human or non-human animal in need of treatment for a pathological condition, disorder, or disease.
  • non-human animal includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc.
  • the subject is a human.
  • the subject is an experimental animal or animal suitable as a disease model.
  • tautomer refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another. It will be apparent to one of ordinary skill in the art that certain compounds of the present invention may exist in tautomeric form. All such tautomeric forms of the compounds are within the scope of this invention.
  • the terms “treat”, “treating”, and “treatment” include: (1) preventing a pathological condition, disorder, or disease, i.e. causing the clinical symptoms of the pathological condition, disorder, or disease not to develop in a subject that may be predisposed to the pathological condition, disorder, or disease but does not yet experience any symptoms of the pathological condition, disorder, or disease; (2) inhibiting the pathological condition, disorder, or disease, i.e. arresting or reducing the development of the pathological condition, disorder, or disease or its clinical symptoms; or (3) relieving the pathological condition, disorder, or disease, i.e. causing regression of the pathological condition, disorder, or disease or its clinical symptoms.
  • Treatment means any manner in which the symptoms of a pathological condition, disorder, or disease are ameliorated or otherwise beneficially altered.
  • the subject in need of such treatment is a mammal, more preferable a human.
  • zinc-binding domain or "zing-binding group" within a compound of the present invention refers to any chemical functionality that is a ligand for transition metals (see, e.g., Cohen et al, 2004, JAm Chem Soc 126(27):8388-8389).
  • Somoza et al. described the crystal structures of human HDAC8 complexed with four structurally diverse hydroxamate inhibitors and deposited the complexes in the Protein Data Bank, e.g., HDAC8 with MS344 (ID code 1T67), HDAC8 with SAHA (ID code 1T69), HDAC8 with TSA (ID code 1T64) and GHDAC8 with CRA-A (ID code IVKG) (Somoza et al, 2004, Structure 12: 1325-1334; incorporated herewith by reference in its entirety).
  • HDAC8 inhibitors The rational design of specific HDAC8 inhibitors is based upon a comparative analysis of the HDAC8, HDAH, and HDLP structures with bound hydroxamate inhibitors (Somoza et al., 2004, Structure 12: 1325-1334). The amino acid sequences and overall active site topology of the enzymes are similar.
  • the catalytic machinery comprising the zinc ion that facilitates amide hydrolysis is found at the bottom of a long, narrow pocket (about 12 A deep; Vannini et al, 2004, Proc Natl Acad Sci USA 101(42): 15064-15069); just above which are the conserved and catalytically important amino acid residues Y306 and H180 (Somoza et al, 2004, Structure 12: 1325-1334).
  • the zinc ion is bound to carboxylate oxygens of D 178 and D267, and to the N ⁇ l atom of Hl 80 (Somoza et al, 2004, Structure 12: 1325-1334).
  • the rim of the pocket is formed by three conserved hydrophobic amino acid residues, F 152, F208, and M274 ( Figure 2). These form the tunnel that the acetyl-lysine substrate and straight-chain hydroxamate inhibitors penetrate to access the catalytic machinery. Similar architecture is found in FIDLP and FIDAH..
  • Vannini et al. noted that immediately below the active site is a tube-like internal cavity filled by several water molecules that could as a shuttle for the reaction product acetate (Vannini et al, 2004, Proc Natl Acad Sci USA 101(42): 15064-15069). There is also a second metal binding site buried in the interior of the HDAC8 protein in the vicinity of the active site, approximately 7 A from the zinc, which is occupied by a sodium ion (Somoza et al., 2004, Structure 12: 1325-1334) or a potassium ion (Vannini et al, 2004, Proc Natl Acad Sci USA 101(42): 15064-15069).
  • HDAC8 structure was solved with four different hydroxamate inhibitors bound.
  • the structure of HDAC8 was used to guide the design of HDAC 8 -selective inhibitors.
  • the active site topology of HDAC8 showed large structural differences depending on which inhibitor was bound to the active site. It is surprisingly malleable. For example in the SAHA:HDAC8 co-crystal structure, the active site is deep and narrow similar to the HDLP and HDAH structures. (Somoza et al, 2004, Structure 12: 1325-1334).
  • CRA-A aryl linker
  • This pocket is created by movement of amino acid residue F 152 away from its normal position packed against amino acid residue M274 to form the lip of the active site tunnel ( Figure 3). This shift may be a consequence of the more sterically demanding aryl hydroxamate CRA-A versus the aliphatic hydroxamate SAHA binding the active site.
  • the compounds of the present invention possess a zinc-binding group linked to a moiety or group of moieties that are positioned to a hydrophilic subpocket between HDAC8 residues methionine 274 (M274) and phenylalanine 152 (F 152) in proximity to the active site zinc ion of the HDAC8 polypeptide.
  • the compounds of this invention can be divided into two groups, one group of which comprises primarily compounds that are known for other applications, but that have not been described as HDAC inhibitors (although this group includes at least one novel compound active for the purpose of this invention), and a second group that comprises novel compounds and that have this property.
  • W represents a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocy
  • X represents a bond, an optionally substituted C 1 -C 3 alkylene group, an optionally substituted C 2 -C 3 alkenylene group or -C ⁇ C-, in which optional substituents are selected from one or more of halogen, hydroxy, lower alkoxy, lower alkylthio, lower haloalkoxy, and lower haloalkylthio; and
  • Y represents a zinc-binding moiety
  • Y represents a hydroxamic acid group or a hydroxamic acid derivative, i.e. a group having the formula in which Ri represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are as defied above in connection with moiety W.
  • Compounds of this group include a number of compounds described by Summers et al. and by Boldt et al. (Summers et al. 1987, J Med Chem 30:574-580; Summers et al. 1990, JMed Chem 33:992-998; BoX ⁇ X et al, 2006, Organic Letters 8: 1729-1732; incorporated by reference in their entirety).
  • Z is -NH, O, or S
  • R 1 represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate,
  • One group of preferred compounds of this type includes those having the formula
  • R 3 and R 4 are independently hydrogen, lower haloalkyl, lower alkyl, — CO 2 H, — NO 2 , lower alkyl carboxylate, lower alkoxy, or — CN.
  • a particularly preferred compound of this group has the above formula in which R 3 and R 4 are both hydrogen, namely
  • R 2 is 1- or 2- naphthyl, which have the formula
  • X oxygen or sulfur and R 3 and R 4 are as defined above.
  • Preferred compounds of this type include those in which X is sulfur or oxygen, respectively and R 3 and R 4 are both hydrogen, namely
  • R 2 is a 1- or 2- naphthyl group, namely
  • FIG. 4 Compounds 1-6 synthesized, purified, characterized and tested for HDAC inhibition as described herein, are depicted in Figure 4.
  • Figure 5 depicts a scheme for the synthesis of HDAC8 inhibitors of the present invention. These inhibitors are aryl hydroxamates like CRA-A, however, lacking the linker domain of CRA-A, and were expected to induce formation of the sub-pocket. Because the typical linker domain is missing in compounds 1-6, the aryl groups are only a short distance from the hydroxamic acid group, the zinc-binding group. Such molecules should be excluded from HDACs that lack the sub-pocket of HDAC8, yet be able to bind and chelate zinc (Zn 2+ ) in HDAC8 due to its enlarged pocket.
  • structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention.
  • structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13 C- or 14 C-enriched carbon are within the scope of this invention.
  • the compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds.
  • the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.
  • the compounds of the present invention may exist as salts.
  • the present invention includes such salts.
  • the present invention provides compounds, which are in a prodrug form.
  • Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention.
  • prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.
  • Certain compounds of the present invention can exist in solvated forms, including hydrated forms, as well as unsolvated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
  • Phenyl-substituted pyrroles (e.g., Compound 7) can be made by reaction of methyl cinnamate or another cinnamate with tosylmethyl isocyanate, followed by hydrolysis of the resulting pyrrole ester and reaction with thionyl chloride (Figure 9A).
  • Phenyl-substituted pyrroles, furans and thiophenes can be made by the reaction of bromothiophenecarboxylic acid or bromofurancarboxylic acid with an aryl boronic acid.
  • Example 5 below depicts such a synthesis.
  • Naphthyl- substituted pyrroles, furans and thiophenes can be made similarly, using a naphthyl boronic acid (Figure 10B):
  • the compounds of the present invention inhibiting an HDAC8 activity were purified using standard laboratory methods and characterized.
  • HDAC Inhibitors are identified using methods known in the art and described herein. A number of different screening protocols can be used to identify compounds that inhibit the expression or activity of an HDAC8.
  • Assays for identifying selective HDAC8 inhibitors versus general inhibitors may be conducted in a cell based or cell free format.
  • an assay may comprise incubating or contacting a subject polypeptide or subject nucleic acid, with a test agent or compound of the present invention under conditions in which a level or an activity of the subject polypeptide or subject nucleic acid can be inhibited, and monitoring or determining the level of inhibition in the presence of the test agent or compound relative to the absence of the test agent or compound.
  • the methods of the present invention described above may optionally comprise the step of determining or detecting a polypeptide, such as an HDAC8 polypeptide.
  • a polypeptide such as an HDAC8 polypeptide.
  • Other polypeptides described herein, such as a histone polypeptide or a tubulin polypeptide or other HDACs can also be determined using the following methods.
  • Determining or detecting a polypeptide may be done in a variety of ways, including, but not limited to detecting the respective polypeptides in a biological sample, a cell, an organ, or in an animal, including human and non-human animals.
  • the expression level of a polypeptide may be determined by a variety of methods, including, but not limited to, affinity capture, mass spectrometry, traditional immunoassays and immunoprecipitation assays, PAGE, Western Blotting, RIA, or HPLC as described herein, or as known by one of skill in the art.
  • Detection paradigms that can be employed to this end include optical methods, electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy.
  • the level of an HDAC8 polypeptide in the absence or presence of a compound can be assessed.
  • Other antibodies specifically detecting a phosphorylated HDAC8 polypeptide are useful for detecting differences in the phosphorylation status of HDAC8 in the absence or presence of a compound.
  • Polypeptides acetylated at lysine residues may be detected using the anti-lysine antibodies described herein. Details are described in Example 8 and Figure 7.
  • HDAC polypeptides for use in the testing a property (e.g., selective inhibition of an HDAC8 polypeptide) of compounds of the present invention and a property of compounds that can be prepared by one of skill in the art using the teachings described herein include naturally occurring HDAC polypeptides and recombinantly expressed HDAC polypeptides.
  • an HDAC8 polypeptide can be a naturally occurring HDAC8 polypeptide or a recombinant HDAC8 polypeptide.
  • a naturally occurring HDAC8 polypeptide can be purified, e.g., from human or mouse tissue or e.g., from human or mouse cells.
  • Recombinant HDAC8 polypeptide can be purified from any suitable expression system as known in the art, e.g., purification of recombinant proteins form a host cell, preferably a mammalian host cell.
  • HDAC8 polypeptides can be purified to substantial purity by standard techniques, e.g., including, but not limited to column chromatography, immunopurification methods, selective precipitation using ammonium sulfate, and others.
  • An HDAC8 polypeptide may be expressed and purified as described, e.g., see Somoza et ah, (Somoza et ah, 2004, Structure 12:1325-1334) who purified an HDAC8 polypeptide from Spodoptera frugiperda SF9 cells or Trichoplusia ni Hi5 cells using a baculovirus expression system.
  • an HDAC8 polypeptide is purified from E.coli as described (Hu et ah, 2000, J Biol Chem 275(20): 15254-64).
  • HDAC8 polypeptide may be expressed and purified with or without an affinity tag, such as an N-terminal poly-histidine affinity tag, FLAG-epitope tag, HA-epitope tag, and the like.
  • HDAC polypeptides useful to practice the present invention include HDAC fusion proteins, HDAC homologs, HDAC isoforms, HDAC orthologs, and in particular HDAC8 fusion proteins, HDAC8 homologs, HDAC8 isoforms, and HDAC8 orthologs.
  • HDAC8 deacetylase activity Various assays have been described to detect HDAC8 deacetylase activity.
  • useful assays are those described by North et al. and Verdin et al, who reported assays for detecting deacetylase activity using, e.g., histones as a substrate (North et al, 2005, Methods 36(4):338-45; Verdin et al, 2004, Methods Enzymol 377: 180-96; incorporated by reference in their entirety).
  • HDAC8 deacetylating activity of a histone substrate can be monitored by, e.g., immunoblotting using an anti histone antibody detecting both acetylated and deacetylated histone and an antibody which is specific for acetylated histone.
  • Example 8 and Figure 7 An additional assay is described in the present invention in Example 8 and Figure 7. This assay involves immunoblotting using anti-tubulin antibodies and anti-acetylated lysine antibodies to detect the acetylation status of tubulin, one of the HDAC8 substrate.
  • the acetylation status of an HDAC8 substrate cane be determined in the absence or presence of a compound of the present invention.
  • a compound of the present invention that inhibits HDAC8 deacetylase activity will result in an increase of the acetylation status of the HDAC8 substrate (see Example 8, Figure 7).
  • Preferred compounds of the invention have an IC50 (inhibition potency or, by definition, the concentration of inhibitor which reduces HDAC8 activity by 50%) of less than about 500 ⁇ M, preferably less than about 100 ⁇ M, more preferably less than about 25 uM, even more preferably less than about 10 ⁇ M and most preferably less than about 1 ⁇ M.
  • IC50 inhibition potency or, by definition, the concentration of inhibitor which reduces HDAC8 activity by 50%
  • Exemplary compounds of the invention are listed in Tables 1 and 2. Table shows inhibition ofHDAC8.
  • a compound of the present invention is a selective inhibitor of HDAC8 when compared
  • the interaction between two molecules, such as HDAC8 and a compound of the present invention or a derivative compound thereof can also be detected, e.g., using a fluorescence assay in which at least one molecule is fluorescently labeled.
  • a fluorescence assay in which at least one molecule is fluorescently labeled.
  • FET or FRET for fluorescence resonance energy transfer includes fluorescence energy transfer (FET or FRET for fluorescence resonance energy transfer) (see, for example, Lakowicz et al, U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No. 4,868,103).
  • a fluorophore label on the first, 'donor' molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy.
  • the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues.
  • Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor.' Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed.
  • a FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter.
  • fluorescence polarization Another example of a fluorescence assay is fluorescence polarization (FP).
  • FP fluorescence polarization
  • determining the ability of a protein to bind to a target molecule or the ability of a compound to bind to a subject polypeptide can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky, 1991, Anal C hem 63:2338-2345; Szabo et ah, 1995, Curr Opin Struct Biol 5:699-705).
  • Biomolecular Interaction Analysis see, e.g., Sjolander and Urbaniczky, 1991, Anal C hem 63:2338-2345; Szabo et ah, 1995, Curr Opin Struct Biol 5:699-705.
  • "Surface plasmon resonance" or "BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore).
  • Binding of HDAC8 inhibitors described herein to an HDAC8 polypeptide can be determined by a variety of methods. One method involves crystallization of the HDAC8 polypeptide in the presence of an HDAC8 inhibitor of the invention as described by Somoza et al. and Vannini et al. (Somoza et al, 2004, Structure 12: 1325-1334; Vannini et al, 2004, Proc Natl Acad Sci USA 101(42): 15064-15069).
  • Example 6 and Figure 6 present exemplary assays and results for determining the selectivity of inhibition of a compound of the present invention.
  • a compound of the present invention inhibits an HDAC8 polypeptide with an efficiency of greater than 5 fold, preferably greater than 10 fold, more preferably greater than 25 fold, even more preferably greater than 50 fold, and most preferably greater than 100 fold over another HDAC polypeptide, such as HDACl or HDAC6.
  • Identification and testing of compounds for inhibiting a level or activity of an HDAC8 and compounds for modulating a level, acetylation status, or activity of a cellular substrate of HDAC8 can be performed using in vitro assays. Exemplary in vitro assays are described herein, and specifically in Example IE.
  • Identification and testing of compounds for inhibiting a level or activity of an HDAC8 and compounds for modulating a level, acetylation status, or activity of a cellular substrate of HDAC8 can also be performed using cell-based assays.
  • An exemplary cell-based assay is described in Examples II and 8 and shown in Figure 7.
  • eukaryotic cells such as mammalian cells are used.
  • yeast cells my be used.
  • the cell can be a primary cell isolated from a donor biological sample.
  • the cell can be an established cell line as made available by the American Type Culture Collection.
  • a cell can also be a cell that is transiently or stably transfected with an expression construct, such as an HDAC8 expression construct.
  • An HDAC8 expression construct comprises an HDAC8 encoding nucleic acid in a vector suitable for expression of HDAC8 in a prokaryotic or eukaryotic cell.
  • the HDAC8 encoding nucleic acid can either encode a full- length HDAC8 polypeptide or a fragment thereof as described herein.
  • Expression control elements such as promoter and enhancer elements are operably linked to the HDAC8 encoding nucleic acid. The making and using of expression construct is within the skill of one of skilled in the art.
  • Identification and testing of a compound for inhibiting a level or activity of an HDAC8 polypeptide can also be performed in vivo.
  • a compound is administered to an animal, preferably a mouse, and blood samples or tissue samples are taken from the animal at various times after administration of the compound and tested for the presence or activity of the HDAC8 polypeptide, a level, acetylation status, or activity of a cellular substrate of HDAC8, such as a histone polypeptide or a tubulin polypeptide.
  • LD50 abbreviation for "Lethal Dose, 50%”
  • median lethal dose of a toxic substance or radiation is the dose required to kill half the members of a tested population, e.g., a mammalian cell or in an animal.
  • LD 50 figures are frequently used as a general indicator of a substance's toxicity.
  • the LD50 is usually expressed as the mass of substance, e.g., a compound of the present invention, administered per unit mass of test subject, such as grams of substance per kilogram of body mass. Stating it this way allows the relative toxicity of different compounds to be compared, and normalizes for the variation in the size of the animals exposed.
  • the LD 50 of a substance is given in milligrams per kilogram of body weight.
  • Lethal dosage often varies depending on the method of administration; for instance, many substances are less toxic when taken by mouth than when intravenously addministered. For this reason, LD 50 figures are often qualified with the mode of administration, e.g. "LD 50 i.v.
  • SARs provide information about the activity of related compounds or agents in at least one relevant assay. Correlations are made between structural features of an agent of interest and an activity. For example, it may be possible by evaluating SARs for a family of agents that interact with an HDAC8 polypeptide to identify one or more structural features required for activity. A library of agents can then be produced that vary these features, and then the library is screened.
  • Structure-based design can include determining a structural model of the physical interaction of the agent and its target, such as an HDAC8 polypeptide. The structural model can indicate how an antagonist of the target can be engineered. Such antagonist may be useful in altering lifespan regulation.
  • pharmacophores are a highly valuable and useful concept in drug discovery and drug-lead optimization.
  • a pharmacophore is defined as a distinct three dimensional (3D) arrangement of chemical groups essential for biological activity. Since a pharmaceutically active molecule must interact with one or more molecular structures within the body of the subject in order to be effective, and the desired functional properties of the molecule are derived from these interactions, each active compound must contain a distinct arrangement of chemical groups which enable this interaction to occur.
  • the chemical groups can be represented by (a) an atom or group of atoms; (b) pseudo- atoms, for example a center of a ring, or the center of mass of a molecule; (c) vectors, for example atomic pairs, electron lone pair directions, or the normal to a plane.
  • a pharmacophore can be used to search a database of chemical compound, e.g., for those having a structure compatible with the pharmacophore (see, for example, U.S. Pat. No. 6,343,257; Martin, 1992, JMedChem 35, 2145-54).
  • Database search queries are based not only on chemical property information but also on precise geometric information.
  • a compound Once a compound is identified that matches the pharmacophore, it can be tested for activity, e.g., for binding to a polypeptide and/or for modulating a biological activity of a polypeptide, e.g., decreasing the enzymatic activity of an HDAC8 polypeptide.
  • a compound is identified that is designed to interact with an HDAC8 polypeptide or binds to an HDAC8 polypeptide by employing a structure of the HDAC8 polypeptide.
  • the HDAC8 is a human HDAC8 (Somoza et al, 2004, Structure 12: 1325-1334; Vannini et al, 2004, Proc Natl Acad Sci USA 101(42): 15064-15069).
  • structures of other mammalian HD AC8 polypeptides may also be used.
  • Three-dimensional structures for potential compounds are generated by entering amino acid or nucleotide sequences or chemical formulas of compounds, as described herein. The three-dimensional structure of the potential compound is then compared to that of HDAC8 to identify binding sites HDAC8. Binding affinity between the HDAC8 polypeptide and compounds is determined using energy terms to determine which compounds have an enhanced probability of binding to the HDAC8 polypeptide.
  • Methods for testing and assaying compounds, agents or antagonists identified by methods described herein, are provided herein and involve a variety of accepted tests to determine whether a given candidate agent, or compound is useful to practice a method of the present invention.
  • Methods of the present invention may optionally comprise the step of detecting a nucleic acid, such as a mRNA or a polypeptide.
  • a method comprises determining or detecting a mRNA, preferably an HDAC8 mRNA or an mRNA encoding a cellular substrate of HDAC8.
  • Other mRNAs encoding polypeptides described herein, such as histone polypeptide or tubulin polypeptide can also be determined using the following methods. Methods of evaluating mRNA expression of a particular gene are well known to those of skill in the art, and include, inter alia, hybridization and amplification based assays.
  • Methods of detecting and/or quantifying the level of a gene transcript (mRNA or cDNA made therefrom) using nucleic acid hybridization techniques are known to those of skill in the art.
  • one method for evaluating the presence, absence, or quantity of a polynucleotide involves a Northern blot.
  • Gene expression levels can also be analyzed by techniques known in the art, e.g., dot blotting, in situ hybridization, RNase protection, probing DNA microchip arrays, and the like (e.g., see Sambrook, J., Fritsch, E. F., and Maniatis, "Molecular Cloning A Laboratory Manual” by T. published by Cold Spring Harbor Laboratory Press, 2nd edition, 1989).
  • amplification-based assays are used to measure the expression level of a gene.
  • the nucleic acid sequences act as a template in an amplification reaction ⁇ e.g., Polymerase Chain Reaction, or PCR).
  • an amplification reaction e.g., Polymerase Chain Reaction, or PCR.
  • the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the level of an mRNA in the sample.
  • Methods of quantitative amplification are well known to those of skill in the art. Detailed protocols for quantitative PCR are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
  • a TaqMan based assay is used to quantify a polynucleotide.
  • TaqMan based assays use a fluorogenic oligonucleotide probe that contains a 5' fluorescent dye and a 3' quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3' end.
  • the 5' nuclease activity of the polymerase e.g., AmpliTaq, results in the cleavage of the TaqMan probe.
  • This cleavage separates the 5' fluorescent dye and the 3' quenching agent, thereby resulting in an increase in fluorescence as a function of amplification ⁇ see, for example, Heid et al., 1996, Genome Res 6(10):986-94; Morris et al., 1996, J Clin Microbiol 34(12):2933-6).
  • LCR ligase chain reaction
  • high throughput screening methods are employed for identifying additional compounds inhibiting a level or activity of an HDAC8 polypeptide.
  • High throughput assays involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds. Such "combinatorial chemical libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity, such as inhibiting a level or activity of an HDCA8 polypeptide.
  • the compounds already identified herein serve as conventional "lead compounds" or can themselves be used as therapeutics.
  • Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art.
  • Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, 1991, Int J Pept Prot Res 37:487- 493 (1991) and Houghton et al, 1991, Nature 354:84-88).
  • Other chemistries for generating chemical diversity libraries can also be used.
  • Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No. WO 93/20242), random bio-oligomers (e.g., PCT Publication No.
  • benzodiazepines e.g., U.S. Pat. No. 5,288,514
  • diversomers such as hydantoins, benzodiazepines and dipeptides
  • vinylogous polypeptides Hagihara et al, 1992, J Amer Chem Soc 114:6568
  • nonpeptidal peptidomimetics with glucose scaffolding Hirschmann et al., 1992, J Amer Chem Soc 114:9217-9218
  • analogous organic syntheses of small compound libraries Chen et al., 1994, J Amer Chem Soc 116:2661
  • oligocarbamates Cho et al., 1993, Science 261 : 1303
  • peptidyl phosphonates Campbell et al., 1994, J Org Chem 59:658
  • High throughput assays are often used in screening for modulators, i.e., identifying inhibitors and activators.
  • identifying inhibitors for HDAC8 polypeptide it is possible to screen up to several thousand different candidate agents or ligands in a single day.
  • each well of a microtiter plate can be used to run a separate assay against a selected potential agent, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single agent.
  • a single standard microtiter plate can assay about 100 (e.g., 96) agents. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different agents. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different agents are possible using the integrated systems of the invention. More recently, microfluidic approaches to reagent manipulation have been developed.
  • the molecule of interest such as an HDAC8 polypeptide
  • the tag can be any of a variety of components.
  • a molecule that binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.
  • tags and tag binders can be used, based upon known molecular interactions well described in the literature.
  • a tag has a natural binder, for example, biotin, protein A, or protein G
  • tag binders avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.
  • Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis Mo).
  • any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair.
  • an appropriate antibody to form a tag/tag binder pair.
  • Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature.
  • the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody.
  • receptor-ligand interactions are also appropriate as tag and tag-binder pairs, such as agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993)).
  • cell membrane receptors e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule
  • toxins and venoms can all interact with various cell receptors.
  • hormones e.g., opiates, steroids, etc.
  • intracellular receptors e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides
  • lectins e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides
  • drugs lectins
  • sugars e.g., nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies
  • nucleic acids both linear and cyclic polymer configurations
  • oligosaccharides oligosaccharides
  • proteins e.g.
  • Synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
  • Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids.
  • polypeptide sequences such as poly gly sequences of between about 5 and 200 amino acids.
  • Such flexible linkers are known to those of skill in the art.
  • poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc., Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
  • Tag binders are fixed to solid substrates using any of a variety of methods currently available.
  • Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder.
  • groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups.
  • Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces.
  • the invention provides in vitro assays for identifying, in a high throughput format, agents that can decrease a level or activity of and F1DAC8 polypeptide.
  • Control reactions that measure a level or activity of HDAC8 in a reaction that does not include a potential inhibitor are optional, as the assays are highly uniform. Such optional control reactions are appropriate and increase the reliability of the assay. Accordingly, in some embodiments, the methods of the invention include such a control reaction.
  • "no inhibitor" control reactions which do not include an inhibitor of HDAC8 provide a background level of binding activity.
  • the compounds identified herein find use in a variety of methods, for example compounds can be used for (i) inhibiting a level or activity of an HDAC8 polypeptide, (ii) modulating a level, acetylation status, or activity of an HDAC8 substrate polypeptide, (iii), or (iii) treatment of a pathological condition, disorder or disease.
  • These methods can be practiced in vitro and in vivo.
  • patients treated by any one of the methods are humans and non-human animals.
  • this invention relates to methods of inhibiting an HDAC8 polypeptide using compounds 1, 2, 3, 4, or 6.
  • the present invention provides a method for inhibiting the enzymatic activity of an HDAC8 polypeptide, i.e., the catalytic activity of histone deacetylation. In a preferred embodiment inhibiting the enzymatic activity of an HDAC8 polypeptide is selective.
  • a level or activity of an HDAC8 polypeptide can be inhibited, i.e., decreased or repressed, in vitro or in vivo.
  • a method for inhibiting a level or an activity of an HDAC8 polypeptide comprises the step of contacting an HDAC8 polypeptide with a compound described herein, wherein the level or activity of the HDAC8 polypeptide is inhibited.
  • Agents identifiable by a screening method of the present invention can also be used to inhibit a level or activity of an HDAC8 polypeptide.
  • the HDAC8 polypeptide may be in a cell, preferably a mammalian cell and more preferred in a human cell.
  • a preferred activity of HDAC8 polypeptide is the deacetylase activity of HDAC8, preferably the deacetylation of a histone.
  • the method of inhibiting a level or an activity of an HDAC8 polypeptide may include the step of determining the level or activity of the HDAC8 polypeptide prior to contacting the HDAC8 polypeptide with a compound of the present invention.
  • the method of inhibiting a level or an activity of an HDAC8 polypeptide may include the step of determining the effect of the compound of the present invention on the level or activity of the HDAC8 polypeptide.
  • the present method provides a method for increasing the acetylation status of a histone polypeptide or a tubulin polypeptide.
  • this method comprises the step of contacting an HDAC8 polypeptide with a compound described herein, wherein the contacting of said compound to the HDAC8 polypeptide in a cell comprising a histone polypeptide or a tubulin polypeptide results in an increase of the acetylation status of the histone polype4ptide or tubulin polypeptide.
  • Assays for determining the acetylation status of an HDAC8 target polypeptide are described herein.
  • HDAC8 Inhibiting The Interaction Between HDAC8 And A Polypeptide
  • Class I HDACs such as HDAC8 play an important role in gene silencing as they are recruited to key locations in nucleosomes through their interactions with transcription complexes. (Ng and Bird, 2000, Trends Biochem Sci 25: 121-128). The interaction of HDAC8 with a transcription complex is indicative of a multiprotein complex forming in a mammalian cell.
  • a method for using a compound of the present invention in the inhibition of a complex formation between an HDAC8 polypeptide and a transcription factor.
  • this method comprises the step of contacting a cell expressing an HDAC8 polypeptide and a transcription factor interacting with an HDAC8 polypeptide with a compound of the present invention, wherein the interaction of HADC8 and a transcription factor is inhibited.
  • the present invention provides a method for modulating a level, acetylation status, or activity of an HDAC8 substrate.
  • a preferred HDAC8 substrate is a histone polypeptide or a tubulin polypeptide.
  • this method comprises the step of contacting a sample comprising an HDAC8 polypeptide and an HDAC8 substrate with a compound described herein, wherein the level, acetylation status, or activity of the HDAC8 substrate is modulated.
  • the HDAC8 polypeptide and the HDAC8 substrate may be in a cell, preferably a mammalian cell and more preferred in a human cell.
  • a preferred activity of an HDAC8 substrate is modulation of gene expression, i.e., increasing or decreasing either alone or in combination with a transcription factor the expression level of a gene of interest.
  • the present invention provides a method for increasing the cellular expression of a repressed HDAC8 target gene.
  • this method comprises the step of contacting a cell comprising an HDAC8 polypeptide and a repressed HDAC8 target gene with a compound described herein, wherein the cellular expression of the repressed HDAC8 target gene is increased.
  • the present invention provides a method for inhibiting unwanted growth, proliferation or survival of a cell.
  • a preferred cell is a cancer cell.
  • this method comprises the step of contacting a cell comprising an HDAC8 polypeptide with a compound described herein, wherein the unwanted growth, proliferation or survival of the cell is inhibited.
  • HDAC 8 polypeptide is an unusual HDAC family member. Recent data suggest that HDAC8 polypeptide is constitutively localized to the cytoplasm and its expression in primary cells is restricted to smooth muscle (Waltregny et al, 2004, Am J Pathol 165:553- 564). Cell fractionation assays performed with primary human smooth muscle cells (HSMCs) showed that HDAC8, in contrast to HDACl and HDAC3, was enriched in cytoskeleton-bound protein fractions and insoluble cell pellets, suggesting an association of HDAC8 with the cytoskeleton.
  • HSMCs primary human smooth muscle cells
  • HDAC8 associates with smooth muscle ⁇ -actin ( ⁇ -SMA), but not with ⁇ -actin (Waltregny et al., 2005, Faseb J 19(8):966-8).
  • RNAi ablation of HDAC8 in these cells by siRNA interference results in a contraction-deficient phenotype, i.e., the capacity of HSMCs to contract collagen lattices was strongly reduced (Waltregny et al, 2005, Faseb J 19:966-968).
  • HDAC8 deacetylates non-histone protein(s) reminiscent of the role of HDAC6 plays in deacetylating tubulin (Hubbert et al, 2002, Nature 417(6887):455-458). Identifying the proteins that are targets of HDAC8 may expand the range of targets and functions of the HDAC family yet further.
  • the present invention provides a method for regulating smooth muscle cell contraction.
  • this method comprises the step of contacting a smooth muscle cell comprising an HDAC8 polypeptide with a compound described herein, wherein the contraction of the smooth muscle cell is regulated.
  • a compound described herein wherein the contraction of the smooth muscle cell is regulated.
  • the method of regulating smooth muscle cell contraction is performed in an animal, a human or a non-human animal.
  • the compounds of the present invention are also useful in methods for the treatment of a pathological condition, disorder, or disease.
  • HDAC8 may play a role in one of the most frequent types of acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • AML results from a chromosomal translocation, Inversion(l ⁇ ), creating an abnormal fusion protein, Invl (Durst et al, 2003, MoI Cell Biol 23 :607-619).
  • the inversion(l ⁇ ) protein product (Invl) associates with HDAC8 into a protein complex and is associated with aberrant, constitutive genetic repression that is thought to cause the disease.
  • the repression that is mediated by this complex is sensitive to HDAC inhibitors (Durst et al, 2003, MoI Cell Biol 23:607-619).
  • the specific HDAC8 inhibitors provided herein are useful for the treatment of AML.
  • a preferred pathological condition, disorder or disease that can be treated according to the present invention is acute myeloid leukemia.
  • disorders related to, associated with or caused (directly or indirectly) by acute myeloid leukemia are also amenable to treatment using a method according to the present invention.
  • the present invention provides a method for the treatment of an individual having acute myeloid leukemia.
  • this method comprises the step of administering to an individual having acute myeloid leukemia a therapeutically effective amount of a compound that inhibits a level or activity of an HD AC8 polypeptide, wherein the individual having acute myeloid leukemia is treated.
  • the method for the treatment of an individual having acute myeloid leukemia comprises the step of administering a pharmaceutical composition to the individual; wherein the pharmaceutical composition comprises a biologically active compound as described herein and a pharmaceutically acceptable carrier wherein the individual having acute myeloid leukemia is treated.
  • HDAC deregulation has been linked to several types of cancer, suggesting a use for HDAC inhibitors in oncology. Treatment with HDAC inhibitors causes tumor cells to cease growth and to either differentiate or become apoptotic. Several small molecules, including SAHA, are currently in clinical trials for oncology indications (Johnstone, 2002, Nat Rev Drug Di scov 1 :287-289).
  • cancer another preferred pathological condition, disorder or disease that can be treated according to the present invention.
  • disorders related to, associated with or caused (directly or indirectly) by cancer are also amenable to treatment using a method according to the present invention.
  • the present invention provides a method for the treatment of an individual having cancer.
  • this method comprises the step of administering to an individual having cancer a therapeutically effective amount of a compound that inhibits a level or activity of an HDAC8 polypeptide, wherein the individual having cancer is treated.
  • the method for the treatment of an individual having cancer comprises the step of administering a pharmaceutical composition to the individual; wherein the pharmaceutical composition comprises a biologically active compound as described herein and a pharmaceutically acceptable carrier, wherein the individual having cancer is treated.
  • HD Huntington's disease
  • R6/2 HD mouse model Recent evidence indicated that transcriptional dysregulation may contribute to the molecular pathogenesis of this disease.
  • Hockly et al. have conducted preclinical trials with SAHA in the R6/2 HD mouse model and reported that SAHA increased histone acetylation in the brain (Hockly et al., 2003, Proc Natl Acad Sci USA, 100:2041-2046). Further, SAHA dramatically improved the motor impairment in R6/2 mice, clearly validating the pursuit of this class of compounds as HD therapeutics (Hockly et al, 2003, Proc Natl Acad Sci USA, 100:2041- 2046).
  • another preferred pathological condition, disorder or disease that can be treated according to the present invention is a neurodegenerative disorder.
  • disorders related to, associated with or caused (directly or indirectly) by a neurodegenerative disorder are also amenable to treatment using a method according to the present invention.
  • a preferred neurodegenerative disorder is Huntington's disease, and amyotrophic lateral sclerosis (Lou Gehrig's).
  • the present invention provides a method for the treatment of an individual having a neurodegenerative disorder.
  • this method comprises the step of administering to an individual having a neurodegenerative disorder a therapeutically effective amount of a compound that inhibits a level or activity of an HD AC8 polypeptide, wherein the individual having the neurodegenerative disorder is treated.
  • the method for the treatment of an individual having a neurodegenerative disorder comprises the step of administering a pharmaceutical composition to the individual, wherein the pharmaceutical composition comprises a biologically active compound as described herein and a pharmaceutically acceptable carrier, wherein the individual having the neurodegenerative disorder is treated.
  • Severe ⁇ -thalassemia (thalassemia major, Cooley anemia) is characterized by insufficient production of adult ⁇ -globin chains with subsequent excess of ⁇ -globin chains leading to ineffective erythropoiesis, intramedullar degradation of erythroid cells, and lifelong transfusion requirement of affected patients.
  • One molecular treatment strategy of this disease comprises the reactivation of fetal ⁇ -globin production to substitute for the lack of ⁇ -globin chains.
  • Witt et al. have investigated the role of HDAC inhibitors for use in stimulating fetal hemoglobin expression in human K562 erythroleukemia cells (Witt et al, 2003, Blood 101 :2001-2007).
  • apicidin (cyclo-[L-(2-amino-8- oxodecanoy ⁇ -L-fN-methoxytryptopha ⁇ -L-isoleucyl-D-pipecolinyl) was the most potent compound compared with other HDAC inhibitors, such as TSA, MS-275, HC-toxin, SAHA and previously tested compounds such as butyrate, phenylbutyrate, isobutyramide, hydroxyurea, 5-aza-cytidine (Witt et al, 2003, Blood 101 :2001-2007). Hyperacetylation of histones correlated with the ability of HDAC inhibitors to stimulate fetal hemoglobin expression (Witt et al, 2003, Blood 101 :2001-2007).
  • Another preferred pathological condition, disorder or disease that can be treated according to the present invention is a genetic disorder.
  • disorders related to, associated with or caused (directly or indirectly) by a genetic disorder are also amenable to treatment using a method according to the present invention.
  • a preferred genetic disorder which can be treated using the compounds of the present invention to inhibit HDAC8, is ⁇ -thalassemia.
  • the present invention provides a method for the treatment of an individual having a genetic disorder.
  • this method comprises the step of administering to an individual having a genetic disorder a therapeutically effective amount of a compound that inhibits a level or activity of an HDAC8 polypeptide, wherein the individual having the genetic disorder is treated.
  • the method for the treatment of an individual having a genetic disorder comprises the step of administering a pharmaceutical composition to the individual, wherein the pharmaceutical composition comprises a biologically active compound as described herein and a pharmaceutically acceptable carrier, wherein the individual having the genetic disorder is treated.
  • RNA interference experiments indicated that several HDACs, including HDACl, HDAC8, and HDAC6 influence beta interferon (IFN-beta) gene expression in response to virus infection (Nusinzon and Horvath, 2006, MoI Cell Biol 26(8):3106-13). While HDACl and HDAC8 repressed IFN-beta expression, HDAC6 acted as a coactivator.
  • IFN-beta beta interferon
  • another preferred pathological condition, disorder or disease that can be treated according to the present invention is a viral infection.
  • disorders related to, associated with or caused (directly or indirectly) by a viral infection are also amenable to treatment using a method according to the present invention.
  • a viral infection that can be treated using the subject method is a viral infection caused by, e.g., Sendai virus and vesicular stomatitis virus (VSV).
  • VSV vesicular stomatitis virus
  • the HDAC8 inhibitors of the present invention may also find use in the reactivation of latent viruses such as varicella-roster virus (VZV), herpes, and human immunodeficiency virus (HIV).
  • the present invention provides a method for the treatment of an individual having a viral infection.
  • this method comprises the step of administering to an individual having a viral infection a therapeutically effective amount of a compound that inhibits a level or activity of an HDAC8 polypeptide, wherein the individual having the viral infection is treated.
  • the method for the treatment of an individual having a viral infection comprises the step of administering a pharmaceutical composition to the individual, wherein the pharmaceutical composition comprises a biologically active compound as described herein and a pharmaceutically acceptable carrier, wherein the individual having the viral infection is treated.
  • Treatment of the viral infection by the subject method can me monitored by detecting, e.g., mRNA levels for beta interferon or beta interferon polypeptides. An increase of mRNA levels for beta interferon or beta interferon polypeptides indicates treatment.
  • the HDAC8 inhibitors of the present invention may also find use in the reactivation of latent viruses such as varicella-roster virus (VZV), herpes, and human immunodeficiency virus (HIV).
  • latent viruses such as varicella-roster virus (VZV), herpes, and human immunodeficiency virus (HIV).
  • the present invention provides a method for the reactivation of a latent virus in a cell.
  • this method comprises the step of contacting a cell with a compound that inhibits a level or activity of an HDAC8 polypeptide, wherein the latent virus in the cell is reactivated.
  • the method for the reactivation of a latent virus is performed in an individual and comprises the step of administering a pharmaceutical composition to the individual, wherein the pharmaceutical composition comprises a biologically active compound as described herein and a pharmaceutically acceptable carrier, wherein the latent virus in the individual is reactivated.
  • Reactivation of a latent virus by the subject method can me monitored by detecting, e.g., mRNA levels for a viral RNA or viral polypeptides. An increase of viral mRNA levels or viral polypeptides indicates reactivation of the latent virus.
  • a compound of the present invention is combined with a methylation inhibitor, such as 5-aza-2'deoxycytidine. Co administration of an HDAC8 inhibitor of the present invention and a methylation inhibitor may lead to a synergistic effect between the HDAC8 inhibitor and the methylation inhibitor (Cameron et al, 1999, Nat Genet 21(1): 103-7).
  • Compounds of the present invention are useful in the manufacture of a pharmaceutical composition or a medicament.
  • a pharmaceutical composition or medicament can be administered to a subject for the treatment of, for example, a pathological condition or disease as described herein.
  • the present invention provides a pharmaceutical composition or a medicament comprising at least a compound as described herein that inhibits the level or activity of an HDAC8 polypeptide and a pharmaceutically acceptable carrier.
  • Compounds and agents of the present invention and compounds and agents identified by a method of the present invention are useful in the manufacture of a pharmaceutical composition or a medicament comprising an effective amount thereof in conjunction or mixture with excipients or carriers suitable for either enteral or parenteral application.
  • a preferred pharmaceutical composition for inhibiting a level or activity of an HDAC8 polypeptide comprises (i) a compound as described herein or a compound obtained or obtainable according to a subject screening method described herein, and (ii) a pharmaceutical acceptable carrier.
  • the compound may be provided in a therapeutically effective dose for use in a method for treatment as described herein.
  • compositions or medicaments for use in the present invention can be formulated by standard techniques using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are described herein and in "Remington's Pharmaceutical Sciences” by E.W. Martin. Compounds and agents of the present invention and their physiologically acceptable salts and solvates can be formulated for administration by any suitable route, including via inhalation, topically, nasally, orally, parenterally, or rectally.
  • the administration of the pharmaceutical composition may be made by intradermal, subdermal, intravenous, intramuscular, intranasal, intracerebral, intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural, intracoronary or intratumoral injection, with a syringe or other devices.
  • Transdermal administration is also contemplated, as are inhalation or aerosol administration. Tablets and capsules can be administered orally, rectally or vaginally.
  • a pharmaceutical composition or a medicament can take the form of, for example, a tablet or a capsule prepared by conventional means with a pharmaceutically acceptable excipient.
  • Tablets may be either film coated or enteric coated according to methods known in the art.
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, for example, suspending agents, for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats; emulsifying agents, for example, lecithin or acacia; non-aqueous vehicles, for example, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils; and preservatives, for example, methyl or propyl-p-hydroxybenzoates or sorbic acid.
  • the preparations can also contain buffer salts, flavoring, coloring, and/or sweetening agents as appropriate. If desired, preparations for oral administration can be suitably formulated to give controlled release of the active compound.
  • compositions for parenteral administration can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative.
  • injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are preferably prepared from fatty emulsions or suspensions.
  • the compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.
  • a suitable vehicle for example, sterile pyrogen-free water
  • they may also contain other therapeutically valuable substances.
  • the compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.
  • the compounds and agents may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • a suitable propellant for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • the dosage unit can be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base, for example, lactose or starch.
  • Suitable formulations for transdermal application include an effective amount of a compound or agent of the present invention with carrier.
  • Preferred carriers include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host.
  • transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.
  • Matrix transdermal formulations may also be used.
  • Suitable formulations for topical application are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.
  • the compounds and agents can also be formulated in rectal compositions, for example, suppositories or retention enemas, for example, containing conventional suppository bases, for example, cocoa butter or other glycerides.
  • the compounds and agents can be formulated as a depot preparation.
  • Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions can, if desired, be presented in a pack or dispenser device that can contain one or more unit dosage forms containing the active ingredient.
  • the pack can, for example, comprise metal or plastic foil, for example, a blister pack.
  • the pack or dispenser device can be accompanied by instructions for administration.
  • a pharmaceutical composition or medicament comprises (i) an effective amount of a compound as described herein that inhibits a level or activity of an HDAC8 polypeptide and (ii) another therapeutic agent.
  • a therapeutic agent may be used individually, sequentially, or in combination with one or more other such therapeutic agents (e.g., a first therapeutic agent, a second therapeutic agent, and a compound of the present invention).
  • Administration may be by the same or different route of administration or together in the same pharmaceutical formulation.
  • the therapeutic agent is a methylation inhibitor, such as 5-aza-2'deoxycytidine.
  • a pharmaceutical composition or medicament is administered to a subject, preferably a human or a non-human animal, at a therapeutically effective dose to prevent, treat, or control a pathological condition or disease as described herein.
  • the pharmaceutical composition or medicament is administered to a subject in an amount sufficient to elicit an effective therapeutic response in the subject.
  • An effective therapeutic response is a response that at least partially arrests or slows the symptoms or complications of the pathological condition, disorder, or disease.
  • An amount adequate to accomplish this is defined as "therapeutically effective dose” also referred to as "therapeutically effective amount.”
  • the dosage of active agents administered is dependent on the species of warmblooded animal (mammal), the body weight, age, individual condition, surface area or volume of the area to be treated and on the form of administration.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse effects that accompany the administration of a particular compound in a particular subject.
  • a unit dosage for oral administration to a mammal of about 50 to 70 kg may contain between about 5 and 500 mg of the active ingredient.
  • a dosage of the active compounds of the present invention is a dosage that is sufficient to achieve the desired effect.
  • Optimal dosing schedules can be calculated from measurements of agent accumulation in the body of a subject. In general, dosage may be given once or more daily, weekly, or monthly. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates.
  • the dosage of active agents administered is also dependent on the nature of the agent.
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • the protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • Exemplary doses of the compounds described herein include milligram or microgram amounts of the compound per kilogram of subject or sample weight (e.g., about 1 microgram per-kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a compound depend upon the potency of the compound with respect to the expression or activity to be modulated.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • a pharmaceutical composition or medicament comprising compounds or agents of the present invention is administered in a daily dose in the range from about 1 mg of each compound per kg of subject weight (1 mg/kg) to about lg/kg for multiple days.
  • the daily dose is a dose in the range of about 5 mg/kg to about 500 mg/kg.
  • the daily dose is about 10 mg/kg to about 250 mg/kg.
  • the daily dose is about 25 mg/kg to about 150 mg/kg.
  • a preferred dose is about 10 mg/kg.
  • the daily dose can be administered once per day or divided into subdoses and administered in multiple doses, e.g., twice, three times, or four times per day.
  • a subject with a therapeutically effective amount of a compound can include a single treatment or, preferably, can include a series of treatments.
  • compounds or agents may be administered for multiple days at the therapeutically effective daily dose.
  • therapeutically effective administration of compounds to treat a pathological condition or disease described herein in a subject requires periodic (e.g., daily) administration that continues for a period ranging from three days to two weeks or longer.
  • agents will be administered for at least three consecutive days, often for at least five consecutive days, more often for at least ten, and sometimes for 20, 30, 40 or more consecutive days. While consecutive daily doses are a preferred route to achieve a therapeutically effective dose, a therapeutically beneficial effect can be achieved even if the agents are not administered daily, so long as the administration is repeated frequently enough to maintain a therapeutically effective concentration of the agents in the subject.
  • Optimum dosages, toxicity, and therapeutic efficacy of such compounds or agents may vary depending on the relative potency of individual compounds or agents and can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio, LD 50 ZED 50 .
  • Agents that exhibit large therapeutic indices are preferred. While agents that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue to minimize potential damage to normal cells and, thereby, reduce side effects.
  • the data obtained from, for example, cell culture assays and animal studies can be used to formulate a dosage range for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (the concentration of the agent that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 concentration of the agent that achieves a half-maximal inhibition of symptoms
  • levels in plasma can be measured, for example, by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the dose equivalent of agents is from about 1 ng/kg to 100 mg/kg for a typical subject.
  • the present invention relates to food, drink or feed with an activity to inhibit a level or activity of an HDAC8 polypeptide.
  • Such food drink or feed can be produced by a general method for producing foods and drinks or feeds, including, adding an active compound as described herein, e.g., a compound that inhibits a level or activity of an HDAC8 polypeptide, to a raw or cooked material of the food, drink or feed.
  • the food, drink or feed in accordance with the present invention can be molded and granulated in the same manner as generally used for foods, drinks or feeds.
  • the concentration of the active compound is preferably 0.001 to 10 % by weight, more preferably 0.01 to 10 % by weight and most preferably 0.1 to 10 % by weight of the food, drink or feed comprising such active agent.
  • Specific foods or drinks include, for example, juices, refreshing drinks, soups, teas, sour milk beverages, dairy products such as fermented milks, ices, butter, cheese, yogurt, processed milk and skim milk, meat products such as ham, sausage, and hamburger, fish meat, cereal, bran, cake products, egg products such as seasoned egg rolls and egg curd, confectioneries such as cookie, jelly, snacks, and chewing gum, breads, noodles, pickles, smoked products, dried fishes, soy sauce-seasoned boiled foods and seasonings.
  • dairy products such as fermented milks, ices, butter, cheese, yogurt, processed milk and skim milk
  • meat products such as ham, sausage, and hamburger, fish meat, cereal, bran, cake products
  • egg products such as seasoned egg rolls and egg curd
  • confectioneries such as cookie, jelly, snacks, and chewing gum, breads, noodles, pickles, smoked products, dried fishes, soy sauce-seasoned boiled foods and seasonings.
  • cyclodextrin includes ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin. Included within “cyclodextrin” are derivatives of cyclodextrin, e.g., ether, ester and amide derivatives and modified cyclodextrins as described in U.S. Patent Nos. 5,134,127 and 6,407,079.
  • Food, drinks and feed with an activity to inhibit a level or activity of an F1DAC8 polypeptide may be further supplemented with a nutritious composition (protein, lipid, saccharide, vitamins and/or mineral).
  • a nutritious composition protein, lipid, saccharide, vitamins and/or mineral.
  • kits are also provided by the present invention.
  • such kits may include any or all of the following: assay reagents, buffers, a compound, agent or compound of the present invention, an HDAC8 polypeptide, a histone polypeptide or any other polypeptide described herein, an HDAC8 nucleic acid, a histone nucleic acid or any other nucleic acid described herein, an anti-HDAC8 antibody, an anti-histone antibody, an anti-acetyl-lysine antibody, or any other antibody described herein, hybridization probes and/or primers detecting an HDAC8 nucleic acid, a histone nucleic acid or any other nucleic acid described herein, an HDACC8 expression construct, a histone expression construct or an expression construct for any other polypeptide described herein, or any other compound or composition described herein.
  • a therapeutic product may include sterile saline or another pharmaceutically acceptable emulsion and suspension base.
  • references to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of the same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which they are presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed.
  • kits for inhibiting a level or activity of an HDAC8 polypeptide comprises a container containing a compound as described herein or a compound obtained or obtainable according to a subject screening method.
  • kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention.
  • the instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g.,
  • Such media may include addresses to internet sites that provide such instructional materials.
  • the kit comprises an instruction for contacting the compound to a mammalian cell for inhibiting a level or activity of an HDAC8 polypeptide.
  • a compound inhibits HDAC8 deacetylase activity.
  • the instruction comprises warnings of possible side effects and drug- drug or drug-food interactions.
  • kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user.
  • the kit is a pharmaceutical kit and comprises a pharmaceutical composition comprising (i) a compound as described herein that inhibits a level or activity of an HDAC8 polypeptide, and (ii) a pharmaceutical acceptable carrier.
  • Pharmaceutical kits optionally comprise an instruction stating that the pharmaceutical composition can or should be used for treating a pathological condition, disorder or disease or any other subject method described herein.
  • kits embodiments of the present invention include optional functional components that would allow one of ordinary skill in the art to perform any of the method variations described herein.
  • HEK293 and HeLa cells were obtained from the American Type Culture Collection and cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 LVmL penicillin, and 100 ⁇ g/mL streptomycin and grown in 5% CO 2 at 37 0 C.
  • HDAC8 polypeptides Purification methods of HDAC8 polypeptides, preparation of HDAC8 polypeptides lystaes, cell fractionation and co-immunoprecipitations are described in North et al, Verdin et al, and Waltregny et al. (North et al, 2005, Methods, 36(4):338-45; Verdin et al, 2004, Methods Enzymol 377: 180-96; Waltregny et al, 2005, Faseb J, 19(6):966-8; incorporated by reference in their entireties).
  • SDS-PAGE and western blotting was performed using standard laboratory methods.
  • Western blots were developed with the ECL detection kit (Amersham Pharmacia Biotech, Piscataway, NJ) or West Supersignal reagent (Pierce).
  • Membranes were either nitrocellulose or polyvinylidene fluoride (PVDF) (Immuno-Blot; Bio-Rad Laboratories).
  • the following antisera were employed: antisera specific for acetyl-lysine (#9441, Cell Signalling Technology, Waltham, MA), for acetyl-tubulin (6-1 IB-I, Abeam, Cambridge, MA), tubulin (B-5-1-2. Abeam, Cambridge, MA), acetyl -Hi stone H4 (06-866, Upstate/Millipore).
  • In vitro deacetylation assay may be performed. Equimolar amounts of purified or recombinant HDAC8 and purified recombinant histone or tubulin are incubated in SDAC deacetylation buffer (50 mM Tris-HCl (pH 9.0), 4 mM MgCl 2 , 50 mM NaCl, 0.5 mM DTT) in the presence or absence of NAD (1 mM), in the presence or absence of nicotinamide (10 mM), in the presence of trichostatin A (500 nM), or a Compound of the present invention for 3 h at 32 0 C.
  • SDAC deacetylation buffer 50 mM Tris-HCl (pH 9.0), 4 mM MgCl 2 , 50 mM NaCl, 0.5 mM DTT
  • HDAC8 Inhibitors E. Acetylated Protein Assay For Characterization Of HDAC8 Inhibitors
  • HEIa cells or HEK293 cells were grown in RPMI medium containing 100 units/mL penicillin G sodium and 100 ⁇ g/mL streptomycin sulfate supplemented with 10% fetal bovine serum at 37°C in a 5% CO 2 atmosphere.
  • log-phase cells were used.
  • Serial dilutions of compounds were prepared and added to cells cultured in 6-well plates. If compounds were dissolved in DMSO, control wells contained the same amount of solvent (0.1% final concentration). Cells were treated with TSA, Compound 2 or Compound 5 for 17 hours.
  • bromothiophenecarboxylic acid (or bromofurancarboxylic acid) 1 is mixed with 1 molar equivalent of aryl boronic acid in ethanol with 2 equivalents of saturated aqueous potassium carbonate under nitrogen.
  • Pd[P(Ph 3 )J 4 is added (0.1 equivalents) and the mixture heated to reflux for 4 hours.
  • Ethanol is then removed under reduced pressure and the residue acidified with 10 % HCl and extracted with EtOAc (3x).
  • the combined organic layers is extracted with saturated aqueous bicarbonate.
  • the bicarbonate is then acidified with 10% HCl, producing a precipitate, which is filtered.
  • Example 6 Compounds 1-6 Inhibit HDAC8 Enzymatic Activity
  • Compounds 1-6 were tested as inhibitors of recombinant HDAC8 using the tritiated histone peptide assay as described herein.
  • the data represented as IC 50 values in Table 1, show that these linkerless, sterically demanding aryl hydroxamates inhibit HDAC8 polypeptide. While some of the compounds are moderately potent in this assay, Compounds 5 and 6 have IC50 values of below 1 ⁇ M.
  • Example 7 Compounds 1, 2, 5 And 6 Are Selective for HDACC8 OverHDACl And HDAC6 [0326] To examine the selectivity of the compounds of the present invention towards
  • HDAC8 some were also tested as inhibitors against other HDAC family members.
  • HDACs from class I (HDACl) and class II (HD AC6) were chosen as representatives of the larger family.
  • the inhibitory activity of Compounds 1, 2, 5, and 6 against HDACl and HDAC6 was tested to determine their selectivity towards HDAC8.
  • immunoprecipitated HDACs 1 and 6 were used in the tritiated histone peptide assay as described herein.
  • the data represented as IC50 values in Table 2, show that all hydroxamates with the linkerless scaffold, as represented by Compounds 1, 2, 5 and 6 are > 100-fold selective for HDAC8 over HDACl.
  • linkerless compounds 1 and 2 are >100-fold selective for HDAC8 over HDAC6 and the linkerless compounds 5 and 6 are 82 and 55 fold selective for HDAC8 over HDAC6, respectively.
  • the less potent compounds may be even more selective, since they present steric bulk in closer proximity to the zinc binding group, potentially clashing with the narrow site seen in HDAH and HDLP.
  • HDAC8 inhibitors described herein The level of selectivity for the HDAC8 inhibitors described herein is adequate for these compounds to be useful as HDAC8-specific inhibitor compounds to examine HDAC8's biological roles, identify its acetylation targets as well as determine the role of HDAC8 in the pathological conditions described herein, including cancer, AML, neurodegenerative dieses, genetic diseases and its role in antiviral responses.
  • the compounds of the present invention represent rationally designed, structure- based inhibitors which are useful for specific inhibition for an individual HDAC family member and a step towards the goal of a series of isoform-specific inhibitors for every HDAC member.
  • the relatively simple structures of the compounds described herein may exploit the active-site malleability of HDAC8 and its unique subpocket, resulting in selective inhibition.
  • HDACs that are insensitive to these compounds also lack the HDAC sub-pocket shown in Figure 3, which may emerge as a structural feature that can be exploited to generate additional selective inhibitors.
  • Chemical tools have played a prominent role in HDAC biology since their discover (Taunton et al, 1996, Science 272:408-411).
  • the compounds described herein are useful tools to study the biological function of HDAC8 in smooth muscle cell contraction, identify its protein targets, as well as serve as lead compounds for the treatment of cancer, in particular AML, neurodegenerative diseases, genetic diseases and in regulating the response to viral infections.

Abstract

La présente invention concerne un échafaudage de nouveaux inhibiteurs de HDAC destiné à utiliser une unique sous-poche du site actif de HDAC8. Ces composés sont basés sur l'inspection de structures cristallines d'HDAC8 liées à divers inhibiteurs, qui ont démontré que le site actif d'HDAC est étonnamment malléable et peut loger des structures d'inhibiteurs qui sont distinctes des structures canoniques de groupe coiffe de lieur de groupe de liaison au zinc de SAHA, TSA et d'inhibiteurs semblables d'HDAC. Certains des nouveaux inhibiteurs basés comparés au nouvel échafaudage sont sélectifs à plus de 100 fois pour l'HDAC8 sur des HADC de classe I et de classe II avec des valeurs IC50 inférieures à 1µM contre l'HADC8. La présente invention concerne un nouveau type d'inhibiteurs d'HADC8 exempts de lieurs et des procédés de traitement d'une condition pathologique mettant en œuvre de tels inhibiteurs. Un traitement de cellules humaines avec les nouveaux inhibiteurs selon la présente invention présentent un motif de protéines hyperacétylées comparé avec le large spectre de TSA inhibiteur d'HADC.
PCT/US2008/054126 2007-02-15 2008-02-15 Inhibiteurs de hdac8 WO2008101186A1 (fr)

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JP5725475B2 (ja) * 2010-01-21 2015-05-27 公立大学法人名古屋市立大学 ヒドロキサム酸誘導体及びそれを用いたｈｄａｃ８阻害剤
WO2011089995A1 (fr) * 2010-01-21 2011-07-28 公立大学法人名古屋市立大学 Dérivé de l'acide hydroxamique et inhibiteur de hdac8 l'utilisant
JP2016512512A (ja) * 2013-03-14 2016-04-28 シーエイチディーアイ ファウンデーション，インコーポレーテッド ヒストンデアセチラーゼ阻害剤及びその組成物と使用方法
US10308596B2 (en) 2013-08-20 2019-06-04 City Of Hope HDAC8 inhibitors for treating cancer
JP2016530273A (ja) * 2013-08-20 2016-09-29 シティ・オブ・ホープＣｉｔｙ ｏｆ Ｈｏｐｅ 癌を治療するためのｈｄａｃ８阻害剤
EP3035924A4 (fr) * 2013-08-20 2017-04-19 City of Hope Inhibiteurs de l'hdac8 pour le traitement du cancer
US11505523B2 (en) 2013-08-20 2022-11-22 City Of Hope HDAC8 inhibitors for treating cancer
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