WO2010008558A1 - Composés et procédés d’induction de l’apoptose par interaction avec la chaperonine cct - Google Patents

Composés et procédés d’induction de l’apoptose par interaction avec la chaperonine cct Download PDF

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WO2010008558A1
WO2010008558A1 PCT/US2009/004106 US2009004106W WO2010008558A1 WO 2010008558 A1 WO2010008558 A1 WO 2010008558A1 US 2009004106 W US2009004106 W US 2009004106W WO 2010008558 A1 WO2010008558 A1 WO 2010008558A1
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cct
aryl
triazolo
carcinomas
compound
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PCT/US2009/004106
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English (en)
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Sui Xiong Cai
Mei Ting Vaillancourt
Gisela Claassen
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Cytovia, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to a method of treating, preventing or ameliorating a disease responsive to induction of the caspase cascade and apoptosis in an animal, comprising administering to the animal a compound which binds to CCT (chaperonin-containing TCP-I (t-complex polypeptide- I)); also termed TRic (TCP-I ring complex) or c-cpn (cytosolic chaperonin).
  • CCT chaperonin-containing TCP-I (t-complex polypeptide- I)
  • TRic TCP-I ring complex
  • c-cpn cytosolic chaperonin
  • the present invention also relates to methods for identifying such CCT interacting compounds that are apoptosis inducers.
  • the invention also relates to the use of biochemical and cell based screening assays to identify CCT interacting compounds that may be administered to animals for treating, preventing or ameliorating a disease responsive to induction of the caspase cascade and a
  • Organisms eliminate unwanted cells by a process variously known as regulated cell death, programmed cell death or apoptosis. Such cell death occurs as a normal aspect of animal development, as well as in tissue homeostasis and aging (Glucksmann, A., Biol. Rev. Cambridge Philos. Soc. 26:59-86 (1951); Glucksmann, A, Archives de Biologie 76:419-437 (1965); Ellis, et al, Dev. 772:591-603 (1991); Vaux, et al, Cell 76:111-119 (1994)). Apoptosis regulates cell number, facilitates morphogenesis, removes harmful or otherwise abnormal cells and eliminates cells that have already performed their function.
  • apoptosis occurs in response to various physiological stresses, such as hypoxia or ischemia (PCT published application WO96/20721).
  • PCT published application WO96/20721 PCT published application WO96/20721.
  • morphological changes shared by cells experiencing regulated cell death including plasma and nuclear membrane blebbing, cell shrinkage (condensation of nucleoplasm and cytoplasm), organelle relocalization and compaction, chromatin condensation and production of apoptotic bodies (membrane enclosed particles containing intracellular material) (Orrenius, S., J. Internal Medicine 237:529-536 (1995)).
  • Apoptosis is achieved through an endogenous mechanism of cellular suicide (Wyllie, A.H., in Cell Death in Biology and Pathology, Bowen and Lockshin, eds., Chapman and Hall (1981), pp. 9-34).
  • a cell activates its internally encoded suicide program as a result of either internal or external signals.
  • the suicide program is executed through the activation of a carefully regulated genetic program (Wyllie, et al, Int. Rev. CyL 68:251 (1980); Ellis, et al, Ann. Rev. Cell Bio. 7:663 (1991)).
  • Apoptotic cells and bodies are usually recognized and cleared by neighboring cells or macrophages before lysis. Because of this clearance mechanism, inflammation is not induced despite the clearance of great numbers of cells (Orrenius, S., J. Internal Medicine 237:529-536 (1995)).
  • caspase family of cysteine proteases comprises 14 different members, and more may be discovered in the future. All known caspases are synthesized as zymogens that require cleavage at an aspartyl residue prior to forming the active enzyme. Thus, caspases are capable of activating other caspases, in the manner of an amplifying cascade.
  • Apoptosis and caspases are thought to be crucial in the development of cancer ⁇ Apoptosis and Cancer Chemotherapy, Hickman and Dive, eds., Humana Press (1999)).
  • cancer cells while containing caspases, lack parts of the molecular machinery that activates the caspase cascade. This makes the cancer cells lose their capacity to undergo cellular suicide and the cells become cancerous.
  • control points are known to exist that represent points for intervention leading to activation.
  • CED-9-BCL-like and CED-3-ICE-like gene family products are intrinsic proteins regulating the decision of a cell to survive or die and executing part of the cell death process itself, respectively (see, Schmitt, et al, Biochem. Cell. Biol. 75:301- 314 (1997)).
  • BCL-like proteins include BCL-xL and BAX-alpha, which appear to function upstream of caspase activation.
  • BCL-xL appears to prevent activation of the apoptotic protease cascade, whereas BAX-alpha accelerates activation of the apoptotic protease cascade.
  • chemotherapeutic drugs can trigger cancer cells to undergo suicide by activating the dormant caspase cascade. This may be a crucial aspect of the mode of action of most, if not all, known anticancer drugs (Los, et al, Blood 90:3118-3129 (1997); Friesen, et al, Nat. Med. 2:51 A (1996)).
  • the mechanism of action of current antineoplastic drugs frequently involves an attack at specific phases of the cell cycle.
  • the cell cycle refers to the stages through which cells normally progress during their lifetime. Normally, cells exist in a resting phase termed G 0 . During multiplication, cells progress to a stage in which DNA synthesis occurs, termed S.
  • Antineoplastic drugs such as cytosine arabinoside, hydroxyurea, 6-mercaptopurine, and methotrexate are S phase specific, whereas antineoplastic drugs, such as vincristine, vinblastine, and paclitaxel are M phase specific.
  • Many slow growing tumors e.g. colon cancers, exist primarily in the G 0 phase, whereas rapidly proliferating normal tissues, for example bone marrow, exist primarily in the S or M phase.
  • a drug like 6-mercaptopurine can cause bone marrow toxicity while remaining ineffective for a slow growing tumor.
  • CCT chaperonin-containing TCP-I (t-complex polypeptide- 1); also termed TRic (TCP-I ring complex) or c-cpn (cytosolic chaperonin) is a double ring hexadecameric chaperonin composed of eight subunit species.
  • CCT plays important functions in maintaining cellular homoeostasis by assisting the folding of many proteins, including the cytoskeletal components actin and tubulin (Dekker C. at al. EMBO J.
  • RNA interference RNA interference
  • RNAi RNA interference
  • Depletion of CCT in well-synchronized HeLa cells causes cell cycle arrest at G2, as demonstrated by a low mitotic index and Cdc2 activity, indicating that CCT plays important roles in mitosis.
  • Complete depletion of PIk 1 in well-synchronized cells also leads to G2 block, suggesting that misfolded Plkl might be responsible for the failure of CCT-depleted cells to enter mitosis and Plkl might be a CCT substrate (MoI Cell Biol. 25:4993- 5010 (2005)).
  • [l,2,4]triazolo[3,4-6][l,3,4]thiadiazines has been identified as potent and highly efficacious activators of the caspase cascade and inducers of apoptosis.
  • the present invention relates to the discovery that apoptosis is induced upon the binding of the 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazines to CCT. Such binding is a starting point for initiating the caspase cascade and apoptosis.
  • the present invention relates to compounds which bind to
  • CCT and induce activation of the caspase cascade and apoptosis; pharmaceutical formulations of these compounds; methods of treating, preventing or ameliorating a disease responsive to induction of the caspase cascade in an animal, comprising administering to the animal such compounds; methods for identifying such CCT binding compounds; and use of homogenous, heterogenous, protein and/or cell based screening assays to identify CCT binding compounds that may be administered to animals for treating, preventing or ameliorating a disease responsive to induction of the caspase cascade.
  • a first embodiment of the invention relates to a method of treating, preventing or ameliorating a disease responsive to induction of the caspase cascade in an animal, comprising administering to the animal a compound which binds to CCT, wherein the compound induces activation of the caspase cascade in the animal and the disease is treated, prevented or ameliorated.
  • the compound is not a 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- 6][l,3,4]thiadiazine or derivative thereof.
  • the disease may be a hyperproliferative disease.
  • the hyperproliferative disease may be a cancer.
  • the cancer may be ⁇ odgkin's disease, non- ⁇ odgkin's lymphomas, acute and chronic lymphocytic leukemias, multiple myeloma, neuroblastoma, breast carcinomas, ovarian carcinomas, lung carcinomas, Wilms 1 tumor, cervical carcinomas, testicular carcinomas, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, bladder carcinomas, chronic granulocytic leukemia, primary brain carcinomas, malignant melanoma, small-cell lung carcinomas, stomach carcinomas, colon carcinomas, malignant pancreatic insulinoma, malignant carcinoid carcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas, acute granulocytic leukemia, hairy cell leukemia, neuroblastom
  • the invention also relates to the discovery that CCT are useful for screening for other apoptotic inducing agents.
  • Such screening can employ CCT with one of its substrates, or a labeled substrate, such as fluorescent labeled or biotin labeled substrates.
  • the invention pertains to a method of identifying potentially therapeutic anticancer compounds comprising: (a) contacting CCT with one or more test compounds; and (b) monitoring whether the one or more test compounds binds to the CCT; wherein compounds which bind the CCT are potentially therapeutic anticancer compounds.
  • the invention also pertains to the use of partially or fully purified CCT which may be used in homogenous or heterogenous binding assays to screen a large number or library of compounds and compositions for their potential ability to induce apoptosis.
  • Those compositions capable of binding to CCT are potentially useful for inducing apoptosis in vivo.
  • CCT is known to mediate folding of many newly synthesized proteins, including the tumor suppressor VHL. It has been reported that CCT binding is specified by two short hydrophobic beta strands in VHL that, upon folding, become buried within the native structure. These two small peptides have been determined to be amino acid 116-119 (LWLF, Box 1) and 148-155 (FANITLPV, Box 2) (MoI Cell. 12:1213-24 (2003)). Using a fluorescent labeled Box 1 or Box 2 molecule, a fluorescence polarization assay similar to what has been developed for Hsp70 (Bioorg Med Chem Lett. 18:3749-51 (2008)) can be set up to find compound that binds to CCT and interfere with its protein folding function.
  • determining whether the compound binds specifically to CCT may comprise a competitive or noncompetitive homogeneous assay.
  • the homogeneous assay may be a fluorescence polarization assay or a radioassay.
  • determining whether the compound binds specifically to CCT may comprise a competitive heterogeneous assay.
  • the heterogeneous assay may be a fluorescence assay, a radioassay or an assay comprising avidin and biotin.
  • the CCT may comprise a detectable label.
  • the label on the CCT may be selected from the group consisting of a fluorescent label, a biotin label or a radiolabel.
  • the 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazines may comprise a detectable label.
  • the label on the 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- 6][l,3,4]thiadiazines may be selected from the group consisting of a fluorescent label, a biotin label or a radiolabel.
  • the CCT substrates may comprise a detectable label.
  • the label on the CCT substrates may be selected from the group consisting of a fluorescent label, a biotin label or a radiolabel.
  • the invention also relates to the use of 3-aryl-6-aryl-7H-
  • the invention pertains to a method of identifying potentially therapeutic anticancer compounds comprising: (a) contacting an antibody to 3-aryl-6-aryl- 7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazines or its derivatives; and (b) determining whether the compound binds to the antibody; wherein compounds which bind the antibody are potentially therapeutic anticancer compounds.
  • the invention also relates to the use of the structures of CCT to design compositions that bind these polypeptides, or to design compositions that activate apoptosis.
  • Apoptosis may be induced by the compounds of the present invention within 3 to 48 hours of introduction to the cell, or administration to an animal. Apoptosis may also be induced by such compounds within 1, 2, 3, 4, 5, 6, 7, 8, or 9 hours. These compounds preferably have a molecular weight ranging from 200 Daltons (g/mole) to 20,000 Daltons (g/mole). The compounds may also have a molecular weight ranging from 500 Daltons to 10,000 Daltons.
  • the invention also relates to a complex, comprising: i) a CCT; and ii) a CCT binding compound.
  • the invention also relates to a detectably labeled 3-aryl-6-aryl-7H-
  • [l,2,4]triazolo[3,4-£][l,3,4]thiadiazines or its derivatives comprising i) 3-aryl- 6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazines or its derivatives; ii) optionally a linker; and iii) a label; wherein said 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-6][l,3,4]thiadiazines or its derivatives is covalently linked to said label optionally via said linker.
  • the linker may be N 5 N-(1, 2- aminoethyl); or N,N-(2- ⁇ 2-[2-(2-aminoethoxy)-ethoxy]-ethoxy ⁇ -aminoethyl); N,N-(2-[2-(2-aminoethoxy)-ethoxy]-aminoethyl).
  • the detectable label may be biotin, a fluorescent label, or a radiolabel.
  • the invention also relates to a composition
  • a composition comprising i) 3-aryl-6-aryl-
  • composition may be prepared by bonding 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazines or its derivatives to said solid phase.
  • CCT4 competition experiment CCT4 protein was incubated with either DMSO, 3-(3-methoxvphenyl)-6-(4-nitrophenyl)-7H-[l,2,4]triazolo[3,4-
  • PLKl protein was immunoprecipitated and analyzed by western blotting to demonstrate equivalent translation and isolation of PLKl protein.
  • b In vitro kinase activity of translated PLKl . Immunoprecipitated PLKl was resuspended in in vitro kinase buffer. Purified casein was added as a substrate with 25 ⁇ M ATP and 10 ⁇ Ci ⁇ - 32 P-ATP. Reactions were incubated at 30 0 C for 30 minutes, then run on 14% SDS-PAGE. The gel was then dried and exposed to film.
  • c Quantitation of casein phosphorylation shown in b using Un-Scan-IT.
  • d Compound does not directly affect PLKl kinase activity.
  • apoptosis is a highly conserved, genetically programmed form of cellular suicide characterized by distinct morphological changes such as cytoskeletal disruption, cell shrinkage, membrane blebbing, nuclear condensation, fragmentation of DNA, and loss of mitochondrial function.
  • a caspase is a cysteine protease of the interleukin- l ⁇ /CED-3 family.
  • the caspase cascade is a sequential activation of at least two caspases, or the activation of caspase activity that behaves as if it involves the sequential activation of at least two caspases.
  • a disease which is "responsive to induction of the caspase cascade” is a disease which may be treated with a CCT binding compound.
  • diseases include hyperproliferative and inflammatory diseases.
  • hyperproliferative diseases include any disease characterized by inappropriate cell proliferation.
  • hyperproliferative diseases include skin diseases such as psoriasis, as well as cancer.
  • Non limiting examples of inflammatory diseases include autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes mellitus, lupus and muscular dystrophy.
  • a cell which expresses a cancer phenotype includes cells which are characteristic of cancer. Such cells may have come from animals manifesting a cancer, from animal bone, tissue or fluid manifesting a cancer, or from cancer cell lines well known in the art.
  • cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells or one in which compounds that activate the caspase cascade have therapeutic use.
  • diseases include, but are not limited to, Hodgkin's disease, non-Hodgkin's lymphomas, acute and chronic lymphocytic leukemias, multiple myeloma, neuroblastoma, breast carcinomas, ovarian carcinomas, lung carcinomas, Wilms 1 tumor, cervical carcinomas, testicular carcinomas, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, bladder carcinomas, chronic granulocytic leukemia, primary brain carcinomas, malignant melanoma, small-cell lung carcinomas, stomach carcinomas, colon carcinomas, malignant pancreatic insulinoma, malignant carcinoid carcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic s
  • an effective amount of a compound for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce, the symptoms associated with the disease. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective. The amount may cure the disease but, typically, is administered in order to ameliorate the disease. Typically, repeated administration is required to achieve the desired amelioration of symptoms.
  • treatment means any manner in which the symptoms of a condition, disorder or disease are ameliorated or otherwise beneficially altered.
  • amelioration of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with administration of the composition.
  • EC 5O refers to a dosage, concentration or amount of a particular compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked or potentiated by the particular compound.
  • a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound.
  • the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes.
  • the prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug.
  • Examples of prodrugs of the compounds of the invention include the simple esters of carboxylic acid containing compounds (e.g. those obtained by condensation with a Ci -4 alcohol according to methods known in the art); esters of hydroxy containing compounds (e.g. those obtained by condensation with a C 1-4 carboxylic acid, C 3-6 dioic acid or anhydride thereof (e.g. succinic and fumaric anhydrides according to methods known in the art); imines of amino containing compounds (e.g. those obtained by condensation with a Ci -4 aldehyde or ketone according to methods known in the art); and acetals and ketals of alcohol containing compounds (e.g.
  • biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions, and mixtures.
  • 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazines or its derivatives as used herein include those compounds represented by Formula I:
  • Ari and Ar 2 independently are optionally substituted aryl or optionally substituted heteroaryl.
  • 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazines or its derivatives as used herein also include such compounds having the above ring structure described in WO2008011045.
  • 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazines or its derivatives as used herein also include the following:
  • polynucleotides also includes splice variants.
  • Splice variants refer to a transcribed RNA in which one or more DNA introns are removed. Hence, the skilled artisan will recognize that any of the polynucleotides described herein may have a splice variant.
  • CCT also include polypeptides encoded by these splice variants.
  • Polynucleotides encoding for CCT may include, but are not limited to, those encoding the amino acid sequence of the CCT described herein by themselves. Polynucleotides encoding for CCT also include those encoding an CCT and additional nucleotide sequences.
  • Additional nucleotide sequences may include, but are not limited to i) nucleic acid sequences which encode an amino acid leader or secretory sequence, such as a pre-, pro- or prepro- protein sequence; ii) non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example— ribosome binding and stability of mRNA; and iii) an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the nucleotide sequence encoding the CCT may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa- histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
  • hexa- histidine provides for convenient purification of the fusion protein.
  • the "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37:767-778 (1984).
  • Polynucleotides which encode for CCT may also comprise polynucleotides which hybridize under stringent hybridization conditions to a portion of the polynucleotides described herein, as described in U.S. Patent No. 6,027,916.
  • a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15, 20, 30, 40, 50, 60 or 70 nucleotides (nt) of the reference polynucleotide. These are useful as diagnostic probes and primers.
  • Polynucleotides are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
  • polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding CCT is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the CCT.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • nucleic acid molecule is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequences described herein can be determined conventionally using known computer programs such as the Bestfit program. Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • a subinducing amount of a substance is an amount that is sufficient to produce a measurable change in caspase cascade activity when used in the method of the present invention and which produces a greater measurable change in caspase cascade activity when used in synergistic combination with a CCT binding compound in the method of the present invention.
  • Label is used herein to refer to any atom or molecule that is detectable and can be attached to a protein or test compound of interest.
  • labels include, but are not limited to, radiolabels, fluorescent labels, phosphorescent labels, chemiluminescent labels and magnetic labels. Any label known in the art can be used in the present invention.
  • homogenous assays refer to assays in which all components are mixed together in the same phase. One example of a homogenous assay is where the components mixed together are all in solution.
  • heterogenous assays refer to assays in which a first component is attached to a solid phase such as a bead or other solid substrate and one or more additional components are in solution.
  • fluorophore or "fluorescent group” means any conventional chemical compound, which when excited by light of suitable wavelength, will emit fluorescence with high quantum yield. See, for example, J. R. Lakowicz in "Principles of Fluorescence Spectroscopy," Plenum Press, 1983. Numerous known fluorophores of a wide variety of structures and characteristics are suitable for use in the practice of this invention. In choosing a fluorophore for fluorescence polarization assays, it is preferred that the lifetime of the fluorophore' s exited state be long enough, relative to the rate of motion of the labeled test compound, to permit measurable loss of polarization following emission.
  • Typical fluorescing compounds which are suitable for use in the present invention, include, for example, rhodamine, substituted rhodamine, fluorescein, fluorescein isothiocyanate, naphthofluorescein, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, and umbelliferone.
  • Other suitable fluorescent groups for use in the present invention include, but are not limited to, those described in U.S. Patent Nos. 4,255,329, 4,668,640 and 5,315,015.
  • reporter molecule is synonymous with the term “reporter compound” and the two terms are used interchangeably.
  • a reporter molecule is a fluorogenic, chromogenic or chemiluminescent substrate that produces a signal such as fluorescence, light absorption within the ultraviolet, visible or infrared spectrum, or light emission, under the influence of the caspase cascade.
  • the reporter molecule may be composed of at least two covalently linked parts.
  • One part is an amino acid sequence which may be recognized by any of the intracellular proteases or peptidases that are produced as a result of caspase cascade activation. This sequence is bonded to an aromatic or conjugated moiety that undergoes a detectable physical change upon its release from all or part of the amino acid sequence.
  • Such moieties include a fluorogenic moiety that fluoresces more strongly after the reporter molecule is hydrolyzed by one of the proteases, a chromogenic moiety that changes its light absorption characteristics after the reporter molecule is hydrolyzed by one of the proteases, or a chemiluminescent moiety that produces light emission after the reporter molecule is hydrolyzed by one of the proteases.
  • the aromatic or conjugated moiety may be linked to a plurality of aminoacid sequences.
  • One type of such a reporter molecule is given by Formula IV:
  • Ri is an N-terminal protecting group such as t-butyloxycarbonyl, acetyl, and benzyloxycarbonyl; each AA independently is a residue of any natural or non- natural ⁇ -amino acid or ⁇ -amino acid, or derivatives of an ⁇ -amino acid or ⁇ - amino acid; n is independently 0-5;
  • R 2 and R 3 are the same or different and are independently hydrogen, alkyl or aryl; and R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
  • R 6 is a blocking group which is not an amino acid or a derivative of an amino acid.
  • R 6 blocking groups include, but are not limited to, an alkyloxycarbonyl group such as methoxycarbonyl, an arylalkyloxycarbonyl group such as benzyloxycarbonyl, a C 2-6 acyl (alkanoyl) group such as acetyl, a carbamyl group such as dimethylcarbamyl, and an alkyl, haloalkyl or aralkyl sulfonyl group such as methanesulfonyl.
  • an alkyloxycarbonyl group such as methoxycarbonyl
  • an arylalkyloxycarbonyl group such as benzyloxycarbonyl
  • a C 2-6 acyl (alkanoyl) group such as acetyl
  • carbamyl group such as dimethylcarbamyl
  • alkyl, haloalkyl or aralkyl sulfonyl group such as methanesulfonyl.
  • Example of reporter molecules which are useful for the screening methods of the present invention include N-(Ac-DEVD)-N'-acetyl-Rhodamine 110 (SEQ ID NO.: 1), N-(Ac-DEVD)-iV-ethoxycarbonyl-Rhodamine 110 (SEQ ID NO.: 1), N-( Ac-DE VD)-JV-hexyloxycarbonyl-Rhodamine 110 (SEQ ID NO.: 1), N-( Ac-DE VD)-N l -octyloxycarbonyl-Rhodamine 110 (SEQ ID NO.: 1). See US6,342,611, US6,335,429 and US6,759,207.
  • fluoro genie reporter molecules useful in the practice of the present invention are disclosed in the following United States patents: 4,336,186; 4,557,862; 4,640,893; 5,208,148; 5,227,487; 5,362,628; 5,443,986; 5,556,992; 5,587,490; 5,605,809; 5,698,411; 5,714,342; 5,733,719; 5,776,720, 5,849,513; 5,871,946; 5,897,992; 5,908,750; 5,976,822.
  • Useful reporter molecules are also described in EP 0285179 Bl; EP 623599 Al; WO 93/04192; WO 93/10461; WO 96/20721; WO 96/36729; WO 98/57664; Ganesh, S. et al, Cytometry 20:334-340 (1995); Haugland, R. and Johnson, L, J. Fluorescence 5:119-127 (1993); Haugland, R, Biotechnic and Histochemistry 70:243-251 (1995); Haugland, R., Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, pp. 28 and 54, 6th Ed. (1996); Holskin, B., et al, Anal. Biochem.
  • One embodiment of the invention relates to compounds which bind to
  • Another embodiment of the invention relates to pharmaceutical formulations of these compounds, and methods of administration of compositions comprising these compounds for preventing, treating or ameliorating a disease responsive to induction of the caspase cascade in an animal.
  • Another embodiment of the invention pertains to a method of treating, preventing or ameliorating a disease in an animal comprising administering to the animal a composition comprising a compound which binds to CCT.
  • the present invention includes a therapeutic method useful to modulate in vivo apoptosis or in vivo neoplastic disease, comprising administering to a subject in need of such treatment an effective amount of one or more CCT binding compounds, or a pharmaceutically acceptable salt or prodrug of CCT binding compound described herein, which functions as a caspase cascade activator and inducer of apoptosis.
  • the present invention also includes a therapeutic method comprising administering to an animal an effective amount of a CCT binding compound, or a pharmaceutically acceptable salt or prodrug of a CCT binding compound, wherein the therapeutic method is useful to treat cancer, which is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells.
  • compositions containing therapeutically effective concentrations of the CCT binding compounds formulated for oral, intravenous, local and topical application are administered to an individual exhibiting the symptoms of one or more of these disorders.
  • the amounts are effective to ameliorate or eliminate one or more symptoms of the disorder.
  • An effective amount of a CCT binding compound for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce, the symptoms associated with the disease.
  • Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective.
  • the amount may cure the disease but, typically, is administered in order to ameliorate the disease. Typically, repeated administration is required to achieve the desired amelioration of symptoms.
  • composition comprising a
  • CCT binding compound or a pharmaceutically acceptable salt of a CCT binding compound described herein, which functions as a caspase cascade activator and inducer of apoptosis in combination with a pharmaceutically acceptable vehicle, is provided.
  • Another embodiment of the present invention is directed to a composition effective to inhibit neoplasia comprising a CCT binding compound, or a pharmaceutically acceptable salt or prodrug of a CCT binding compound described herein, which functions as a caspase cascade activator and inducer of apoptosis, in combination with at least one known cancer chemotherapeutic agent, or a pharmaceutically acceptable salt of the agent.
  • alkylating agents such as busulfan, cis- platin, mitomycin C, and carboplatin
  • antimitotic agents such as colchicine, vinblastine, paclitaxel,
  • anti-cancer agents which can be used for combination therapy, include arsenic trioxide, gamcitabine, melphalan, chlorambucil, cyclophosamide, ifosfamide, vincristine, mitoguazone, epirubicin, aclarubicin, bleomycin, mitoxantrone, elliptinium, fludarabine, octreotide, retinoic acid, tamoxifen and alanosine.
  • the CCT binding compound of the invention may be administered together with the at least one known chemotherapeutic agent as part of a unitary pharmaceutical composition.
  • the CCT binding compound of the invention may be administered apart from the at least one known cancer chemotherapeutic agent.
  • the CCT binding compound of the invention and the at least one known cancer chemotherapeutic agent are administered substantially simultaneously, i.e. the CCT binding compounds are administered at the same time or one after the other, so long as the CCT binding compound reach therapeutic levels for a period of time in the blood.
  • Another embodiment of the present invention is directed to compositions and methods effective to inhibit neoplasia comprising a bioconjugate of a CCT binding compound described herein, which functions as a caspase cascade activator and inducer of apoptosis, in bioconjugation with at least one known therapeutically useful antibody, such as Herceptin ® or Rituxan ® , growth factors, such as DGF, NGF; cytokines, such as IL-2, IL-4, or any molecule that binds to the cell surface.
  • the antibodies and other molecules will deliver a CCT binding compound described herein to its targets and make it an effective anticancer agent.
  • bioconjugates could also enhance the anticancer effect of therapeutically useful antibodies, such as Herceptin ® or Rituxan ® .
  • another embodiment of the present invention is directed to compositions and methods effective to inhibit neoplasia comprising a CCT binding compound, or a pharmaceutically acceptable salt or prodrug of a CCT binding compound described herein, which functions as a caspase cascade activator and inducer of apoptosis, in combination with radiation therapy.
  • the CCT binding compound of the invention may be administered at the same time as the radiation therapy is administered or at a different time.
  • Yet another embodiment of the present invention is directed to compositions and methods effective for post-surgical treatment of cancer, comprising a CCT binding compound, or a pharmaceutically acceptable salt or prodrug of a CCT binding compound described herein, which functions as a caspase cascade activator and inducer of apoptosis.
  • the invention also relates to a method of treating cancer by surgically removing the cancer and then treating the animal with one of the pharmaceutical compositions described herein.
  • compositions within the scope of this invention include all compositions wherein the CCT binding compounds of the present invention are contained in an amount which is effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.
  • the CCT binding compounds may be administered to mammals, e.g. humans, orally at a dose of 0.0025 to 100 mg/kg, or an equivalent amount of the pharmaceutically acceptable salt thereof, per day of the body weight of the mammal being treated for apoptosis-mediated disorders.
  • the CCT binding compounds may be administered to mammals, e.g.
  • a dose of 0.025 to 200 mg/kg, or an equivalent amount of the pharmaceutically acceptable salt thereof, per day of the body weight of the mammal being treated for apoptosis- mediated disorders Preferably, approximately 0.01 to approximately 50 mg/kg is orally administered to treat or prevent such disorders.
  • the dose is generally approximately one-half of the oral dose.
  • a suitable intramuscular dose would be approximately 0.0025 to approximately 50 mg/kg, and most preferably, from approximately 0.01 to approximately 10 mg/kg.
  • a known cancer chemotherapeutic agent is also administered, it is administered in an amount which is effective to achieve its intended purpose. The amounts of such known cancer chemotherapeutic agents effective for cancer are well known to those of skill in the art.
  • the unit oral dose may comprise from approximately 0.01 to approximately 50 mg, preferably approximately 0.1 to approximately 10 mg of the CCT binding compound of the invention.
  • the unit dose may be administered one or more times daily as one or more tablets, each containing from approximately 0.1 to approximately 10, conveniently approximately 0.25 to 50 mg of the CCT binding compound or its solvates.
  • the CCT binding compound may be present at a concentration of approximately 0.01 to 100 mg per gram of carrier.
  • the CCT binding compounds of the invention may be administered as part of a pharmaceutical preparation containing suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the CCT binding compounds into preparations that can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the CCT binding compounds into preparations that can be used pharmaceutically.
  • the preparations particularly those preparations, which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations, which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from approximately 0.01 to 99 percent, preferably from approximately 0.25 to 75 percent of active CCT binding compound(s), together with the excipient.
  • nontoxic pharmaceutically acceptable salts of the CCT binding compounds of the present invention are included within the scope of the present invention.
  • Acid addition salts are formed by mixing a solution of the particular apoptosis inducer of the present invention with a solution of a pharmaceutically acceptable non-toxic acid, such as hydrochloric acid, hydrobromic acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, lactic acid, tartaric acid, carbonic acid, phosphoric acid, sulfuric acid, oxalic acid, and the like.
  • Basic salts are formed by mixing a solution of the particular apoptosis inducer of the present invention with a solution of a pharmaceutically acceptable non-toxic base, such as sodium hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate, Tris, N-methyl- glucamine and the like.
  • a pharmaceutically acceptable non-toxic base such as sodium hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate, Tris, N-methyl- glucamine and the like.
  • compositions of the invention may be administered to any animal, which may experience the beneficial effects of the CCT binding compounds of the invention.
  • animals e.g., mammals, e.g., humans and veterinary animals, although the invention is not intended to be so limited.
  • compositions of the present invention may be administered by any means that achieve their intended purpose.
  • administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal or topical routes.
  • administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • compositions of the present invention are manufactured in a manner, which is itself known, e.g., by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes.
  • pharmaceutical preparations for oral use can be obtained by combining the active CCT binding compounds with solid excipients, optionally grinding the resultant mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular: fillers, such as saccharides, e.g. lactose or sucrose, mannitol or sorbitol; cellulose preparations and/or calcium phosphates, e.g. tricalcium phosphate or calcium hydrogen phosphate; as well as binders, such as starch paste, using, e.g. maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone.
  • fillers such as saccharides, e.g. lactose or sucrose, mannitol or sorbitol
  • cellulose preparations and/or calcium phosphates e.g. tricalcium phosphate or calcium hydrogen phosphate
  • binders such as starch paste, using, e.g. maize starch, wheat starch, rice starch, potato starch
  • disintegrating agents may be added, such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Auxiliaries are, above all, flow-regulating agents and lubricants, e.g. silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol.
  • Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices.
  • concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropymethyl-cellulose phthalate, are used.
  • Dye stuffs or pigments may be added to the tablets or dragee coatings, e.g., for identification or in order to characterize combinations of active CCT binding compound doses.
  • Other pharmaceutical preparations which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active CCT binding compounds in the form of granules, which may be mixed with fillers, such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active CCT binding compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin.
  • stabilizers may be added.
  • Possible pharmaceutical preparations which can be used rectally include, e.g. suppositories, which consist of a combination of one or more of the active CCT binding compounds with a suppository base.
  • Suitable suppository bases are, e.g. natural or synthetic triglycerides, or paraffin hydrocarbons.
  • gelatin rectal capsules which consist of a combination of the active CCT binding compounds with a base.
  • Possible base materials include, e.g. liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
  • Suitable formulations for parenteral administration include aqueous solutions of the active CCT binding compounds in water-soluble form, e.g. water-soluble salts and alkaline solutions.
  • suspensions of the active CCT binding compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, e.g. sesame oil; or synthetic fatty acid esters, e.g. ethyl oleate or triglycerides or polyethylene glycol-400 (the CCT binding compounds may be soluble in PEG-400).
  • Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension include, e.g. sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • CCT binding compounds of the invention are employed in topical and parenteral formulations and are used for the treatment of skin cancer.
  • the topical compositions of this invention are formulated preferably as oils, creams, lotions, ointments and the like by choice of appropriate carriers.
  • Suitable carriers include vegetable or mineral oils, white petrolatum (white soft paraffin), branched chain fats or oils, animal fats and high molecular weight alcohol (greater than C 12 ).
  • the preferred carriers are those in which the active ingredient is soluble.
  • Emulsifiers, stabilizers, humectants and antioxidants may also be included as well as agents imparting color or fragrance, if desired.
  • transdermal penetration enhancers can be employed in these topical formulations. Examples of such enhancers can be found in U.S. Patent Nos. 3,989,816 and 4,444,762.
  • Creams are preferably formulated from a mixture of mineral oil, self- emulsifying beeswax and water in which mixture the active ingredient, dissolved in a small amount of an oil such as almond oil, is admixed.
  • a typical example of such a cream is one which includes approximately 40 parts water, approximately 20 parts beeswax, approximately 40 parts mineral oil, and approximately 1 part almond oil.
  • Ointments may be formulated by mixing a solution of the active ingredient in a vegetable oil, such as almond oil with warm soft paraffin and allowing the mixture to cool.
  • a vegetable oil such as almond oil
  • a typical example of such an ointment is one which includes approximately 30% almond oil and approximately 70% white soft paraffin by weight.
  • enteric coating is used herein to refer to any coating over an oral pharmaceutical dosage form that inhibits dissolution of the active ingredient in acidic media, but dissolves rapidly in neutral to alkaline media and has good stability to long-term storage.
  • the dosage form having an enteric coating may also comprise a water soluble separating layer between the enteric coating and the core.
  • the core of the enterically coated dosage form comprises a CCT binding compound.
  • the core also comprises pharmaceutical additives and/or excipients.
  • the separating layer may be a water soluble inert CCT binding compound or polymer for film coating applications.
  • the separating layer is applied over the core by any conventional coating technique known to one of ordinary skill in the art. Examples of separating layers include, but are not limited to sugars, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropyl cellulose, polyvinyl acetal diethylaminoacetate and hydroxypropyl methylcellulose.
  • the enteric coating is applied over the separating layer by any conventional coating technique.
  • enteric coatings include, but are not limited to cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate, carboxymethylethylcellulose, copolymers of methacrylic acid and methacrylic acid methyl esters, such as Eudragit ® L 12,5 or Eudragit ® L 100 (Rohm Pharma), water based dispersions such as Aquateric ® (FMC Corporation), Eudragit ® L 100-55 (Rohm Pharma) and Coating CE 5142 (BASF), and those containing water soluble plasticizers such as Citroflex ® (Pfizer).
  • the final dosage form is either an enteric coated tablet, capsule or pellet.
  • Non-limiting examples of CCT include T-complex protein 1 subunit alpha (TCP-I -alpha) (CCT-alpha) (NCBI Accession No. P17987)(SEQ ID NO.: 2); CCT2 [Homo sapiens] (NCBI Accession No. CAG33352)(SEQ ID NO.: 3); chaperonin containing TCPl, subunit 3 isoform a [Homo sapiens] (NCBI Accession No. NP_005989)(SEQ ID NO.: 4) ; chaperonin containing TCPl, subunit 4 (delta) [Homo sapiens] (NCBI Accession No.
  • NP 006421 (SEQ ID NO.: 5); chaperonin containing TCPl, subunit 5 (epsilon) [Homo sapiens] (NCBI Accession No. NP_036205)(SEQ ID NO.: 6); T-complex protein 1 subunit zeta (TCP-1-zeta) (CCT-zeta) (CCT-zeta-1) (Tcp20) (HTR3) (Acute morphine dependence-related protein 2) (NCBI Accession No.
  • T-complex protein 1 subunit eta TCP-I -eta
  • CCT-eta HV-I Nef-interacting protein
  • chaperonin containing TCPl, subunit 8 theta [Homo sapiens] (NCBI Accession No. NP_006576)(SEQ ID NO.: 9).
  • Non-limiting examples of nucleotide sequences which encode for CCT include Homo sapiens t-complex 1, mRNA (cDNA clone MGC:2234 IMAGE:3349237), complete cds (NCBI Accession No. BC000665)(SEQ ID NO.: 10); Homo sapiens chaperonin containing TCPl, subunit 2 (beta) (CCT2), mRNA (NCBI Accession No.
  • NM_006431 Homo sapiens chaperonin containing TCPl, subunit 3 (gamma), mRNA (cDNA clone MGC:2378 MAGE:2820063), complete cds (NCBI Accession No. BC006501)(SEQ ID NO.: 12); Homo sapiens chaperonin containing TCPl, subunit 4 (delta) (CCT4), mRNA (NCBI Accession No. NM_006430)(SEQ ID NO.: 13); Homo sapiens chaperonin containing TCPl, subunit 5 (epsilon) (CCT5), mRNA (NCBI Accession No.
  • NM_012073 (SEQ ID NO.: 14); Homo sapiens chaperonin containing TCPl, subunit 6 A (zeta 1) (CCT6A), transcript variant 1, mRNA (NCBI Accession No. NM_001762)(SEQ ID NO.: 15) ; Homo sapiens chaperonin containing TCPl, subunit 7 (eta), mRNA (cDNA clone MGC:110985 MAGE:6202511), complete cds (NCBI Accession No. BC088351)(SEQ ID NO.: 16); Homo sapiens chaperonin containing TCPl, subunit 8 (theta) (CCT8), mRNA (NCBI Accession No. NM_006585)(SEQ ID NO.: 17)
  • nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced nucleotide molecule.
  • the actual sequence can be more precisely determined by other approaches including manual nucleotide sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • nucleotides encoding CCT may include splice variants of the nucleotides described herein. See, for example Evans, P. and Kemp, J., "Exon/intron structure of the human transferrin receptor gene," Gene, 199: 123-31 (1997).
  • the present invention also relates to vectors which include the isolated nucleotide molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of CCT by recombinant techniques.
  • CCT may be extracted from cultures of the below described transfected cells and used for the homogenous and heterogenous assays described herein. Alternatively, CCT can be synthesized for these assays using peptide synthetic techniques known in the art. Also, the below described expression vectors and transfected cells are useful for whole cell assays described herein.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged cationic lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors may include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • Vectors which may be used in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH 16a, pNH 18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
  • Introduction of polynucleotides into the host cell can be affected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). Methods of formulating nucleotides with compositions (e.g., lipids) to facilitate introduction of the nucleotide into the cell are disclosed in, for example, U.S. Pat. Nos.
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • a fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins.
  • EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof.
  • CCT can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, or hydroxylapatite chromatography.
  • High performance liquid chromatography HPLC
  • Useful polypeptides include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
  • polypeptides may be glycosylated or may be non-glycosylated.
  • polypeptides may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • One aspect of the present invention relates to a method of identifying
  • CCT binding compounds using homogenous or heterogenous binding assays. This may be accomplished by using non-competitive binding assays, or assays in which test compounds compete with 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- &][l,3,4]thiadiazines or derivatives such as those described in WO2008011045, or the compounds described in this application, such as radioactive labeled 3-aryl-6-aryl-7H-[l ,2,4]triazolo[3,4-6][l ,3,4]thiadiazines analog in Example 3. Any method known to one of ordinary skill in the art that detects binding between a test compound and a protein or antibody may be used in the present invention.
  • test compounds capable of binding to CCT are candidates for activators of apoptosis.
  • Test compounds may be capable of binding to CCT as strongly or more strongly than 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-6][l,3,4]thiadiazines or derivatives.
  • Another aspect of the present invention relates to a method of identifying CCT binding compounds using antibodies to 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-&][l,3,4]thiadiazines or derivatives. Such a method relates to detecting binding between i) an antibody to 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-&][l,3,4]thiadiazines or derivatives, and ii) a test compound.
  • 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazines or derivatives bind CCT
  • an antibody which is specific for 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-6][l,3,4]thiadiazines or derivatives is likely to be specific for other compositions having the physical characteristics that afford CCT specific binding.
  • antibodies can be used to screen chemical libraries for other compositions that bind CCT and that activate apoptosis. In such assays, the antibody may give rise to a detectable signal upon binding a test compound.
  • the antibodies may be labeled with a fluorophore.
  • Antibodies bound to a test compound may also be detected using radiolabels.
  • Assays for use in the present invention are preferably high throughput screening methods, capable of screening large numbers of compounds in a rapid fashion. This includes, for example, screening methods that use microbeads or plates having multiple wells.
  • any homogeneous assay well known in the art can be used in the present invention to determine binding between test compounds of interest and CCT.
  • radioassays, fluorescence polarization assays and time- resolved fluorescence assays may all be used.
  • the assay may be a non-competitive binding assay in which the ability of test compounds to bind the CCT is determined.
  • the assay may be a competitive binding assay where the ability of a test compound to displace the CCT-bound 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazines or derivatives is determined.
  • a homogeneous binding assay used in the present invention may employ fluorescently labeled 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- 6][l,3,4]thiadiazines or derivatives, or fluorescently labeled CCT. Any method known to one of ordinary skill in the art can be used to link the fluorophore to 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazines or derivatives, or polypeptide of interest. See, e.g., Richard P. ⁇ augland, Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals 1992-1994 (5th edit, 1994, Molecular Probes, Inc.).
  • the polarization is directly related to the molecular volume or size of the fluorophore.
  • the polarization value is a dimensionless number (being a ratio of vertical and horizontal fluorescent intensities) and is not affected by the intensity of the fluorophore.
  • instruments for use in the invention include, but are limited to, the Sentry-FP ® fluorescence polarization instrument (Diachemix Corp., Milwaukee, WI); the BEACON ® 2000 fluorescence polarization instrument (PanVera, Madison, WI); the POLARSCAN ® portable fluorescence polarization system (Associates of Cape Cod, Inc., Falmouth, MA); the VICTOR series instruments (PerkinElmer, Inc., Wellesley, MA); and the AFFINTY ® and SYMMETRY ® fluorescence systems (CRi, Inc., Woborn, MA).
  • One embodiment of the invention relates to a non-competitive fluorescent assay.
  • Such an assay employs CCT covalently attached to a fluorophore. Free CCT has higher fluorescence intensity than CCT bound to a test compound. Confer Hwang, et al, Biochemistry 57:11536-11545 (1992). Once the test compound/CCT complex is formed, it rotates and tumbles more slowly and has less fluorescence intensity. Confer "Introduction to Fluorescence Polarization," Pan Vera Corp., Madison, WI, June 17, 1996; Perrin, J. Phys. Rad. 7:390-401 (1926). Hence, when the test compound and CCT bind, the fluorescence intensity of the labeled CCT decreases proportional to binding.
  • a solution of the labeled CCT is prepared and its fluorescence polarization is measured.
  • the CCT and the test compound are mixed together and the solution is allowed to reach equilibrium over some time period.
  • the fluorescence of any test compound/CCT complex which forms is then measured.
  • the decrease in fluorescence intensity is proportional to binding.
  • the test compound binding may be compared to a baseline fluorescence intensity value determined for 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-6][l,3,4]thiadiazines or derivatives bound to CCT.
  • Test compounds that bind to CCT are considered candidates for activators of apoptosis.
  • the skilled artisan will recognize that a variety of parameters such as temperature, time, concentration and pH can be varied to study the binding between the test compound and CCT.
  • the baseline fluorescence polarization value is determined by preparing labeled CCT and measuring its fluorescence polarization.
  • 3-Aryl-6- aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazines or derivatives is mixed with labeled CCT and allowed to equilibrate for a sufficient time to form a complex between 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazines or derivatives and CCT.
  • the fluorescence polarization of the solution comprising the complex is measured.
  • the relative change in the fluorescence polarization is the baseline value against which all other test compounds will be measured.
  • a variety of parameters such as temperature, time, concentration and p ⁇ can be varied to develop a range of values for the change in fluorescence polarization under a variety of conditions.
  • test compounds that bind as strongly as or more strongly than 3- aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazines or derivatives are candidates for activators of apoptosis.
  • [l,2,4]triazolo[3,4-6][l,3,4]thiadiazines or derivatives is first labeled with a fluorophore.
  • An assay similar to a ⁇ SP90 fluorescent polarization assay using labeled geldanamycin can be developed (J Biomol Screen. 9:375-81 (2004)).
  • the labeled 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazines or derivatives is mixed with CCT in a buffered solution.
  • the mixture is allowed to equilibrate and the fluorescence polarization of the 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-&][l,3,4]thiadiazine /CCT (or 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-6][l,3,4]thiadiazines derivative/CCT) complex is measured.
  • the test compound is then introduced into the mixture and allowed to equilibrate.
  • the fluorophore (covalently attached to the 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- 6][l,3,4]thiadiazine) is no longer associated with the bulky CCT, it gives rise to a more intense fluorescence polarization signal. Accordingly, in this embodiment, increases in fluorescent signals are proportional to the ability of a test compound to bind CCT.
  • several components of the mixture can affect the fluorescence intensity other than the labeled moiety.
  • the polarity of the solvent and non-specific binding molecules can have significant affects on the intensity, which can be incorrectly interpreted.
  • an alternative assay for determining test compound/CCT binding for use in the present invention relies on time-resolved fluorescence techniques, which minimizes the above problems.
  • the method of time-resolved fluorescence is described in detail in I. ⁇ emmila, et al, "High Throughput Screening. The Discovery of Bioactive Substances," Chapter 20, J. P. Devlin, ed., Marcel Dekker, Inc., New York (1997).
  • the excited state lifetime of the test compound/CCT complex is longer than that for the impurities and other components that add background fluorescence. Therefore, the solution comprising the test compound/CCT complex mixture may be illuminated and after a short period of time on the order of nano to micro seconds, the solution fluorescence is measured.
  • the fluorescent signal is generated when CCT and 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- 6][l,3,4]thiadiazine or derivative bind.
  • either the CCT or 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-&][l,3,4]thiadiazines is covalently bound to an energy donating Eu-cryptate having a long-lived fluorescent excited state.
  • the other is attached to an energy-accepting protein, allophycocyanin, having a short fluorescent excited state.
  • 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-b][l,3,4]thiadiazine or derivative is radiolabeled and allowed to equilibrate with CCT in solution. Then, a test compound is introduced into the solution and allowed to equilibrate. The CCT (bound either to radiolabeled 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-b][l,3,4]thiadiazine or derivative or to the test compound) is then separated from unbound 3-aryl- 6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative and unbound test compound.
  • test compound is a poor CCT binder
  • most of the CCT will be bound to radiolabeled 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- 6][l,3,4]thiadiazine or derivative and this can be detected by a scintillation counter, photoradiography, or other techniques well known in the art.
  • test compound is a strong CCT binder and displaces radiolabeled 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazine or derivative, then most of the CCT will not be bound to radiolabeled 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-b][l,3,4]thiadiazine or derivative.
  • ability of a test compound to bind CCT is inversely proportional to the amount of radiolabel detected with the CCT.
  • CCT is known to mediate folding of many newly synthesized proteins.
  • CCT protein substrates can be labeled with a fluorescent label, and the interaction of CCT with this substrate and the formation of CCT-substrate complex can be measured by fluorescence polarization.
  • Compound interacts with CCT for example, by binding to the ATP binding site of CCT, can interfere with the function of CCT and the formation of the CCT-substrate complex or the folding of the substrate, and this interference can be measured by the fluorescence polarization.
  • the fluorescent labeled CCT protein substrates can be prepared use the protein as a whole, or fragments of the protein which are known to be substrate of CCT, such as the two short hydrophobic beta strands in VHL (LWLF, Box 1) and 148-155 (FANITLPV, Box 2) that are known to be key for interating with CCT (MoI Cell. 12:1213-24 (2003)).
  • Polo-like kinase 1 is a known substrate of CCT folding (MoI
  • PLKl is in vitro translated. PLKl is then immunoprecipitated from the in vitro translation reaction. The isolated PLKl is used in a kinase assay with casein as a substrate to show Polo-like kinase activity, which indicates that the PLKl is functional and has been properly folded by CCT.
  • This assay can be modified and used to determine whether a compound interacts with CCT and interferes with its protein folding function.
  • PLKl is in vitro translated in the (a) absence or (b) presence of test compound.
  • PLKl in (a) and (b) is used in a kinase assay with casein as a substrate to monitor Polo-like kinase activity, and PLKl protein levels from (a) and (b) are measured to demonstrate that there is equivalent amounts of PLKl synthesized in (a) and (b). If the PLKl kinase activity from (b) is 2-fold less than that of (a), the test compound is determined to interact with CCT and interfere with its protein folding function.
  • Actin is another known substrate of CCT folding and it has been demonstrated that only folded actin will bind to DNase I (Cell 69: 1043-1050 (1992)).
  • actin is in vitro translated. This in vitro translated mixture is then incubated with DNase I conjugated to Sepharose beads. The beads are washed, and the amount of bound actin is assessed. Equivalent translation of actin is shown in parallel with a portion of the mixture from the in vitro translation reaction.
  • This assay can be modified and used to determine whether a compound interacts with CCT and interferes with its protein folding function. In this assay, actin is in vitro translated in the (a) absence or (b) presence of test compound.
  • the in vitro translated mixture in (a) and (b) is used in a binding assay with DNase I conjugated to Sepharose beads, and equivalent translation of actin is shown in parallel with a portion of the mixture from the in vitro translation reaction of (a) and (b). If the amount of bound actin from (b) is 2-fold less than that of (a), the test compound is determined to interact with CCT and interfere with its protein folding function.
  • Detection of the test compound binding to CCT may also be accomplished using heterogeneous assays.
  • Heterogeneous assays for use in the present invention may be based on radioassays, fluorescence-based assays and biotin-avidin based assays.
  • a first component is attached to a solid phase such as a bead or other solid substrate and one or more additional components are in solution.
  • CCT may be bound to a bead or other solid substrate and labeled 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative is introduced as a solution.
  • the label may be a radiolabel, chemiluminescent label, fluorescent label, chromogenic label, or other label well known in the art.
  • separation can occur simply by rinsing the solid substrate with water or a buffer to remove any solution containing unbound labeled 3-aryl-6-aryl- 7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative or unbound test compound.
  • the extent to which CCT remains associated with the detectably labeled 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative is measured. Such measurements can be performed while the CCT remains bound to the bead or solid substrate. Alternatively, such measurements can be made after the CCT has been removed from the bead or solid substrate.
  • decreases in signal associated with the detectable label are proportionally related to increases in the ability of test compounds to bind the CCT by displacing 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative.
  • 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazine or derivative may also be the component bound to the beads or solid substrate.
  • labeled CCT is introduced as a solution and allowed to equilibrate forming the 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative/CCT complexes.
  • the label may be a radiolabel, chemiluminescent label, fluorescent label, chromogenic label, or other label well known in the art. Then, a test compound is added as a solution.
  • Solid phase supports for use in the present invention include any insoluble support known in the art that is capable of binding CCT or 3-aryl-6- aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative.
  • This includes, for example, glass and natural and synthetic polymers such as agaroses, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, and magnetite.
  • the support material may have virtually any possible structural configuration so long as the support-bound molecule is capable of binding to a test compound, 3-aryl-6- aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative, or to the CCT.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod, or hemishperical surface such as the well of a microtitre plate.
  • the surface may be flat such as a sheet, test strip, etc.
  • Radioassay An example of a heterogeneous assay for use in the present invention is the radioassay.
  • a good description of a radioassay may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. S., et al., North Holland Publishing Company, NY (1978), with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T. Examples of other competitive radioassays are given in U.S. Patent Nos. 3,937,799; 4,102,455; 4,333,918 and 6,071,705. Inherent in such assays is the need to separate the bead or substrate bound component from the solution component.
  • separation can include filtering, centrifuging, washing, or draining the solid substrate to insure efficient separation of the substrate bound and solution phases.
  • the radioactive isotope or radiolabel can be detected by such means as the use of a gamma counter or a scintillation counter or by audioradiography.
  • Isotopes which are particularly useful for the purpose of the present invention are: 3 H, 123 I, 125 I, 131 1, 35 S, 32 P, 33 P 5 14 C, 111 In, 97 Ru, 67 Cu, 67 Ga, 68 Ga 5 72 As, 89 Zr and 201 Tl.
  • suitable labels which may be employed in accordance with the present invention.
  • one or more hydrogen and/or carbon atoms of the CCT, 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-£][l,3,4]thiadiazine or derivative are replaced by 3 H and 1 4 C, by methods well known in the art.
  • the CCT is attached to a solid support. Radiolabeled 3-aryl-6-aryl-7H-[ 1 ,2,4]triazolo[3,4-6][l ,3,4]thiadiazine or derivative is prepared.
  • the bound CCT is admixed with the solution comprising radiolabeled 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazine or derivative.
  • the mixture is allowed to equilibrate for a time period.
  • a test compound is added to the mixture and allowed to equilibrate for some time period.
  • the test compound competes for the binding site of the CCT with the radiolabeled 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazine or derivative.
  • the solid support that has bound CCT is removed from the mixture. The amount of radio label associated with the CCT is measured.
  • Decreases in the amount of radiolabel are proportional to the ability of a test compound to displace 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazine or derivative and bind the CCT.
  • the radiation of the solution comprising unbound and uncomplexed radiolabeled 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative can be measured.
  • test compounds that bind to the CCT as strongly or more strongly than 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative can easily be discovered.
  • Alternative labels for use in the heterogeneous assays of the present invention include chemi luminescent labels, such as those described in U.S. Patent No. 4,380,580; and enzyme substrate labels, such as those assays described in U.S. Patent No. 4,492,751.
  • chemi luminescent labels such as those described in U.S. Patent No. 4,380,580
  • enzyme substrate labels such as those assays described in U.S. Patent No. 4,492,751.
  • a fluorescent label may be used.
  • a solution of fluorescently labeled 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- 6][l,3,4]thiadiazine or derivative is prepared.
  • CCT is attached to a solid support.
  • the bound CCT is admixed with the solution comprising fluorescently labeled 3-aryl-6-aryl-7H-[ 1 ,2,4]triazolo[3,4-£][ 1 ,3,4]thiadiazine or derivative.
  • the mixture is allowed to equilibrate for a time period.
  • a test compound is added to the mixture and the mixture is allowed to equilibrate for some time period.
  • the test compound competes for the binding of the CCT with fluorescently labeled 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazine or derivative.
  • the solid support that has bound the CCT is removed from the mixture.
  • the amount of fluorescence associated with the CCT attributed to the fluorescent label is measured. Decreases in the amount of this fluorescence are proportional to the ability of a test compound to displace 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazine or derivative and bind the CCT.
  • the fluorescence of the solution comprising unbound and uncomplexed fluorescently labeled 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative can be measured.
  • test compounds that bind to CCT as strongly or more strongly than 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative can easily be discovered.
  • An alternative heterogeneous assay for use in the present invention is a biotin/avidin based assay.
  • this assay can be performed in the present invention, see, e.g., Blake, R. C, et al. Anal. Biochem. 272:123-134 (1999); Cho, ⁇ . C, et al. Anal. Sciences 75:343- 347 (1999); Choi, M. ⁇ ., et al. Bull. Korean Chem. Soc. 22:417-420 (2001); U.S. Patent No. 6,096,508; U.S. Patent No. 4,863,876; and U.S. Patent No. 4,228,237.
  • avidin may be labeled with any label, preferably, avidin is fluorescently labeled or conjugated to an enzyme.
  • Any detectably labeled enzyme can be used in the present invention, specific examples include, but are not limited to, horseradish peroxidase, alkaline phophatase, ⁇ -galactosidase and glucose oxidase.
  • One particular embodiment of the invention employs a competitive heterogeneous biotin-avidin assay.
  • the test compound competes with 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative for the CCT binding sites.
  • biotinylated 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- 6][l,3,4]thiadiazine or derivative is prepared.
  • the CCT bound to solid support is admixed with the biotinylated 3-aryl-6-aryl-7//-[l,2,4]triazolo[3,4- 6][l,3,4]thiadiazine or derivative and incubated for some defined period of time.
  • 3-Aryl-6-aryl-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative binds to CCT and forms a complex on the solid support.
  • the solid support comprising biotinylated 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-
  • the avidin binds only to the biotinylated 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-b][l,3,4]thiadiazine or derivative.
  • the mixture is allowed to incubate for some defined period of time, and the amount of biotin-avidin complex is measured.
  • the decrease in amount of biotin-avidin complex is directly related to the increase in test compound binding.
  • Test compounds that bind CCT are candidates as apoptosis inducers.
  • the ability of a test compound to effectively compete with 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazine or derivative to bind to the CCT can be ascertained by using base line values.
  • a given assay may be done with labeled 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazine or derivative.
  • the amount of signal associated with that label found in the labeled 3-aryl-6-aryl- 7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazine or derivative bound CCT component can be determined to give a base line value. Then, the test compound may be introduced and a second measurement of the signal attributable to the detectable label is taken which can be compared to the base line value. The extent to which the test compound decreases the base line value is a function of the ability of the test compound to bind CCT.
  • new candidate drugs that induce apoptosis may be identified by assaying for binding between test compounds of interest and antibodies raised against 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- b][l,3,4]thiadiazine or derivative.
  • Antibodies to 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4-b][l,3,4]thiadiazine or derivative may be generated and purified using conventional, well-known methods. Such methods are described for example, in Cohler & Milstein, Nature, 256, pp. 495-497 (1975); "Antibodies-A Laboratory Manual", E. Harlow & D. Lane, Coldspring Harbor Laboratory, pp. 55-144 (1988); C. Williams & M. Chase, in "Methods in Immunology & Immunochemistry," Academic Press, New York, Vol. 1, Chap. 3, (1967); and S. Burchiel, in "Methods in Enzymology,” Vol.
  • an immunogen comprising 3-aryl-6-aryl-7H- [l,2,4]triazolo[3,4-b][l,3,4]thiadiazine or derivative is administered to an animal in order to elicit an immune response against the immunogen.
  • Polyclonal antibodies generated against the immunogen are obtained from the animal antisera and are then purified using well-known methods.
  • Monoclonal antibodies against the immunogen can be obtained from hybridoma cells using well-known methods.
  • Suitable immunogens for raising polyclonal antibodies include, but are not limited to, bioconjugates of 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- b][l,3,4]thiadiazine or derivative.
  • bioconjugates include, but are not limited to, conjugates between 3-aryl-6-aryl-7H-[l,2,4]triazolo[3,4- b][l,3,4]thiadiazine or derivative and any biological molecule, such as proteins, growth factors and cytokines. Examples include, but are not limited to proteins such as bovine hemoglobin; bovine serum albumin; growth factors such as DGF and NGF; and cytokines such as IL-2 and IL-4.
  • Bioconjugates are prepared by any method known to one of ordinary skill in the art. See for example, F. J. Burrows and P. E. Thorpe, "Eradication of large solid tumors in mice with an immunotoxin directed against tumor vasculature," Proc. Natl. Acad. Sd. USA 90:8996-9000 (1993); M. Adamczyk, et ah, "Characterization of Protein-Hapten Conjugates. 2. Electrospray Mass Spectrometry of Bovine Serum Albumin-Hapten Conjugates," Bioconjugate Chem. 7:475-481 (1996); R. B.
  • Sheep, goats and mice can be immunized with the above bioconjugates and antisera can be obtained by methods well known in the art.
  • the antibodies may then be detectably labeled, e.g. with a radiolabel, fluorescence label, enzyme label, biotin, avidin or other label, as described above or according to methods well known in the art. Detection of binding between the test compounds of interest and the antibodies can be done by the homogenous or heterogenous methods as described above, or by any method known in the art.
  • the level of CCT expression can be down regulated through the use of antisense nucleotides.
  • An antisense nucleotide is a nucleic acid molecule that interferes with the function of DNA and/or RNA. This may result in suppression of expression.
  • Antisense oligonucleotides also include any natural or modified oligonucleotide or chemical entity that binds specifically to a pre- mRNA or mature mRNA which results in interference or inhibition with translation of the mature mRNA or prevents the synthesis of the polypeptide encoded by the mature mRNA.
  • Antisense RNA sequences have been described as naturally occurring biological inhibitors of gene expression in both prokaryotes (Mizuno, T., Chou, M-Y, and Inouye, M. (1984), Proc. Natl. Acad. Sci. USA 81, (1966- 1970)) and eukaryotes (Heywood, S. M. Nucleic Acids Res. , 14, 6771-6772 (1986) and these sequences presumably function by hybridizing to complementary mRNA sequences, resulting in hybridization arrest of translation (Paterson, B. M., Roberts, B. E., and Kuff, E. L., (1977) Proc. Natl. Acad. Sci.
  • Antisense oligodeoxynucleotides are short synthetic nucleotide sequences formulated to be complementary to a specific gene or RNA message. Through the binding of these oligomers to a target DNA or mRNA sequence, transcription or translation of the gene can be selectively blocked and the disease process generated by that gene can be halted.
  • the cytoplasmic location of mRNA provides a target considered to be readily accessible to antisense oligodeoxynucleotides entering the cell; hence much of the work in the field has focused on RNA as a target.
  • antisense therapy relies on the ability of antisense oligonucleotides to be taken up by cells and form a stable heteroduplex with the target DNA or mRNA.
  • the end result of antisense oligonucleotide hybridization is the down regulation of the targeted protein's synthesis.
  • Down regulation of protein synthesis by antisense oligonucleotides has been postulated to result from two possible mechanisms: 1) "hybrid arrest," where direct blocking in pre-mRNA and/or mRNA of sequences important for processing or translation prevents full-length proteins from being synthesized; and 2) an RNase H mediated cleavage and subsequent degradation of the RNA portion of the RNA:DNA heteroduplex (Haeuptle, M.
  • Antisense oligonucleotides include S-oligos (nucleoside phosphorothioates) which are isoelectronic analogs of an oligonucleotide (O- oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom.
  • S-oligos may be prepared by treatment of the corresponding O-oligos with 3H- 1 ,2-benzodithiol-3 -one- 1,1 -dioxide which is a sulfur transfer reagent. See Iyer, R.P. et al., J. Org. Chem. 55:4693-4698 (1990) ; and Iyer, R. P. et al., J. Am.
  • Antisense oligonucleotides also include such derivatives as described in U.S. Patent Nos. 6,031,086, 5,929,226, 5,886,165, 5,693,773, 6,054,439, 5,919,772, 5,985,558, 5,595,096, 5,916,807, 5,885,970, 5,877,309, 5,681,944, 5,602,240, 5,596,091, 5,506,212, 5,521,302, 5,541,307, 5,510,476, 5,514,787, 5,543,507, 5,512,438, 5,510,239, 5,514,577, 5,519,134, 5,554,746, 5,276,019, 5,286,717, 5,264,423, as well as WO96/35706, WO96/32474, WO96/29337 (thiono triester modified antisense oligodeoxynucleotide phosphorothioates), WO94/17093 (
  • useful antisense oligonucleotides include derivatives such as S- oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press (1989) which can be prepared, e.g., as described by Iyer et al. (J.
  • Antisense oligonucleotides may be coadministered with an agent which enhances the uptake of the antisense molecule by the cells.
  • the antisense oligonucleotide may be combined with a lipophilic cationic compound which may be in the form of liposomes.
  • the antisense oligonucleotide may be combined with a lipophilic carrier such as any one of a number of sterols including cholesterol, cholate and deoxycholic acid.
  • the antisense oligonucleotide may be conjugated to a peptide that is ingested by cells.
  • useful peptides include peptide hormones, cell surface receptor ligands, antigens or antibodies, and peptide toxins. By choosing a peptide that is selectively taken up by the cells, specific delivery of the antisense agent may be effected.
  • the antisense oligonucleotide may be covalently bound via the 5'H group by formation of an activated aminoalkyl derivative. The peptide of choice may then be covalently attached to the activated antisense oligonucleotide via an amino and sulfhydryl reactive hetero bifunctional reagent.
  • the peptidyl antisense agent Upon exposure of cells to the antisense oligonucleotide bound to the peptide, the peptidyl antisense agent is endocytosed and the antisense oligonucleotide binds to the target CCT mRNA to inhibit translation. See PCT Application Publication No. PCT/US89/02363.
  • the antisense oligonucleotide may be at least a 15-mer that is complementary to a nucleotide molecule coding for CCT as described herein.
  • the antisense oligonucleotides of the present invention may be prepared according to any of the methods that are well known to those of ordinary skill in the art.
  • the antisense oligonucleotides may be prepared by solid phase synthesis. See, Goodchild, J., Bioconjugate Chemistry, 1 :165-167 (1990), for a review of the chemical synthesis of oligonucleotides.
  • the antisense oligonucleotides can be obtained from a number of companies which specialize in the custom synthesis of oligonucleotides.
  • Methods within the scope of this invention include those wherein the antisense oligonucleotide is used in an amount which is effective to achieve inhibition of CCT expression in cells. Determination of effective amounts of each component is within the skill of the art.
  • RNA Interference (RNAi) Mediated Down Regulation of CCT
  • RNAi interfering RNA
  • RNA that results in catalytic degradation of specific mRNAs can also be used to lower gene expression. See U.S. Patent Nos. 6,458,382, 6,506,559 and 6,511,824.
  • complementary sense and antisense RNAs derived from a portion of a gene of interest are synthesized in vitro using techniques well known in the art.
  • the resulting sense and antisense RNAs are annealed in a buffer, and the double stranded RNA is introduced into the cell.
  • RNAi is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene.
  • dsRNA double-stranded RNA
  • Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and mammals are known in the art (Fire A, et al, Nature 397:806-811 (1998); Fire, A., Trends Genet. 15:358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond, S. M., et al., Nature Rev. Genet.
  • RNAi-mediated inhibition of gene expression refers to the absence (or observable decrease) in the level of protein and/or mRNA product from a target gene. Specificity refers to the ability to inhibit the target gene without manifest effects on other genes of the cell.
  • RNAi-mediated inhibition in a cell line gene expression is conveniently assayed by use of a reporter or drug resistance gene whose protein product is easily assayed.
  • Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof.
  • AHAS acetohydroxyacid synthase
  • AP alkaline phosphatase
  • LacZ beta galactosidase
  • GUS beta glucoronidase
  • CAT chloramphenicol acetyltransferase
  • GFP green fluorescent protein
  • HRP horseradish peroxidase
  • Luc nopaline synthase
  • OCS octopine synthase
  • Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.
  • RNAi mediated down regulation is affected by double stranded RNA sequences identical to a portion of the target.
  • double strand RNA sequences comprise a first strand that encodes CCT as described herein and a second strand complementary to the first strand.
  • the double strand RNA comprises a first strand identical to the nucleotides described herein and a second strand complementary to the first strand.
  • an RNA sequence is identical to a DNA sequence even though i) the ribose portion is not deoxyribose as in DNA, and ii) the nucleotide pyrimidine base thymine (usually found in DNA) is replaced by uracil.
  • the double-stranded structure may also be formed by a single self- complementary RNA strand.
  • the double stranded RNA can have insertions, deletions, and single point mutations relative to the target sequence.
  • sequence identity may optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). In one embodiment there is more than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene.
  • the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 0 C or 70 0 C hybridization for 12-16 hours; followed by washing).
  • the length of the identical nucleotide sequences may be at least 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000 or more bases. 100% sequence identity between the RNA and the target gene is not required.
  • the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
  • RNA may include modifications which are well known in the art to either the phosphate-sugar backbone or the nucleosides.
  • the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. Modifications in RNA structure may be tailored to allow specific genetic inhibition.
  • bases may be modified to block the activity of adenosine deaminase.
  • RNA may be produced enzymatically or by partial/total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis.
  • the invention relates to a method for identifying potentially therapeutically effective antineoplastic compounds wherein a test compound is determined to have potential therapeutic efficacy if said caspase cascade activity is enhanced in response to the presence of said test compound, the method comprising (a) obtaining viable cultured eukaryotic cells expressing CCT (and optionally expresses a cancer phenotype) by culturing those cells in a cell growth medium under conditions which result in growth; (b) exposing the viable cultured cells to a test compound for a predetermined period of time at a predetermined temperature; (c) adding a reporter compound having at least one measurable property which is responsive to the caspase cascade; (d) measuring the caspase cascade activity of said exposed viable cultured cells by measuring said at least one measurable property of said reporter compound; and (e) wherein an increase in the measured caspase cascade activity in the presence of the test compound is an indication that the test compound is a potentially therapeutically effective antineoplastic compound.
  • a test compound In contrast to screening methodology using reporter compounds, the ability of a test compound to activate apoptosis can be monitored by microscopically observing changes in cellular morphology.
  • cells can, in conjunction with the screening techniques described above, be assayed for apoptotic morphology using standard techniques well known to those of skill in the art.
  • characteristics of apoptotic morphology are cellular condensation, nuclear condensation, including chromatin condensation, and the apoptotic characteristic plasma membrane ruffling and blebbing referred to as "zeiosis" See Sanderson, C. J., 1982, in Mechanisms of Cell-Mediated Cytotoxicity, Clark, W. R.
  • morphologic changes characteristic of nuclear apoptosis can be assayed and quantified by staining using a DNA-specif ⁇ c fluorochrome such as bis-benzimide (Hoechst-33258; Sigma according to standard methods. See Bose, et al., 1995, Cell 82:405- 414.
  • a DNA-specif ⁇ c fluorochrome such as bis-benzimide (Hoechst-33258; Sigma according to standard methods. See Bose, et al., 1995, Cell 82:405- 414.
  • DNA fragmentation is another morphological change indicative of apoptosis.
  • DNA fragmentation may be detected with the terminal transferase assay (TUNEL; Thiry M., 1992, Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues; J. Histochem. Cytochem. 40:419-441; Gavrieli Y, Sherman Y and Ben-Sasson SA; 1992, Identification of programmed cell death in situ-via specific labeling of nuclear DNA fragmentation; J. Cell Biol. 7/9:493-501).
  • TUNEL terminal transferase assay
  • the TUNEL assay is used to detect 3'OH termini of nicked or broken DNA strands. These nicks or breaks may be generated directly by activating apoptosis. In vivo, apoptosis can be assayed via, for example, DNA terminal transferase nick-end translation, or TUNEL assay, according to standard techniques. See Fuks, Z. et al, 1995, Cancer J. 7:62-72.
  • the present invention relates to a screening method for identifying potentially therapeutically effective antineoplastic compounds by determining the ability of test compounds to alter cellular morphology in cultured eukaryotic cells expressing CCT wherein a test compound is determined to have potential therapeutic efficacy if the cellular morphology is altered in response to the presence of said test compound, the method comprising (a) obtaining cultured eukaryotic cells expressing CCT (and optionally expresses a cancer phenotype) by culturing those cells in a cell growth medium under conditions which result in growth; (b) exposing the viable cultured cells to a test compound for a predetermined period of time at a predetermined temperature; (c) microscopically examining the cellular morphology; and (d) wherein morphological changes indicative of apoptosis in the presence of the test compound is an indication that the test compound is a potentially therapeutically effective antineoplastic compound.
  • a first population expresses an elevated level of CCT relative to a second population.
  • the second population of cells can be normal cells or cells which down regulate CCT (mediated, for example, by antisense nucleotides, RNAi, or altered genes).
  • the first population of cells are normal cells
  • the second population of cells can be cells which down regulate CCT.
  • the first and second population are separately exposed to the test compound and the reporter molecule which gives rise to a measurable property upon activation of the caspase cascade. Any increase in the reporter compound's measurable property in the first population relative to the second population is an indication that the test compound binds CCT, activates the caspase cascade, and is a potentially therapeutic antineoplastic compound.
  • Such a screening method relates to a method of identifying potentially therapeutically effective antineoplastic compounds by determining the ability of test compounds to inhibit cellular culture growth in eukaryotic cells expressing an CCT wherein a test compound is determined to have potential therapeutic efficacy if the cellular culture growth is inhibited in response to the presence of said test compound, the method comprising (a) obtaining cultured eukaryotic cells expressing the CCT (and optionally expresses a cancer phenotype) by culturing those cells in a cell growth medium under conditions which result in growth; (b) exposing the cultured cells to a test compound for a predetermined period of time at a predetermined temperature; (c) following the rate of culture growth; and (d) wherein a decrease in culture growth rate in the presence of the test compound is an indication that the
  • the invention also relates to a method for assaying the potency of a potentially therapeutically effective antineoplastic compound that functions as an activator of the caspase cascade in viable cultured eukaryotic cells having an intact cell membrane and expressing an CCT comprising: (a) obtaining a first and a second population of viable cultured eukaryotic cells, each of which has an intact cell membrane and each of which expresses the CCT (and optionally expresses a cancer phenotype), by culturing said eukaryotic cells in a cell growth medium under conditions which result in growth; (b) exposing the first population to a predetermined amount of a test compound for a predetermined period of time at a predetermined temperature; (c) exposing the second population to an amount of solvent that was used to dissolve the test compound for the predetermined period of time at the predetermined temperature; (a) obtaining a first and a second population of viable cultured eukaryotic cells, each of which has an intact cell membrane and each of which expresses
  • side-by-side screening can be modified to accommodate the above described screening methodologies which utilize microscopic observations of changes in cellular morphology, cell cycle or observations of cellular culture growth rate. Because these modified assays do not follow caspase cascade activation, they do not require addition of a reporter compound.
  • the caspase cascade activity measured for test compounds by this method can also be compared to that measured for compounds which are known to affect enzymes involved in the apoptosis cascade to generate a measure of the relative effectiveness of the test substance.
  • Compounds that can be used in comparison include known activators of enzymes involved in the apoptosis cascade.
  • Known activators, either by direct or indirect mechanisms, of enzymes involved in the apoptosis cascade include but are not limited to vinblastine, etoposide (Yoon, H. J., et al., Biochim. Biophys. Acta. 1395:110-120 (1998)) and doxorubicin (Gamen, S., et al, FEBS Lett. 417:360-364 (1997)) which are topoisomerase II inhibitors; cisplatin (Maldonado et al, Mutat. Res. 381:61-15 (1997)); chlorambucil (Hickman, J.A., Cancer Metastasis Rev. /7:121-139 (1992)) which is an alkylating agent; and fluorouracil, an RNA/DNA anti-metabolite (Hickman, J.A., Cancer Metastasis Rev. 77:121-139 (1992)).
  • a plurality of viable cultured cells are exposed separately to a plurality of test compounds, e.g. in separate wells of a microtiter plate.
  • a large number of test compounds may be screened at the same time.
  • the invention relates to a method for assaying the potency of a test compound to synergise with other cancer chemotherapeutic agents as an activator of the caspase cascade, comprising (a) obtaining a first and a second population of viable cultured eukaryotic cells, each of which has an intact cell membrane and expresses CCT (and optionally expresses a cancer phenotype), by culturing the cell populations in a cell growth medium under conditions which result in growth; (b) exposing the first population to a combination of a predetermined amount of a test compound and a subinducing amount of a known cancer chemotherapeutic agent for a first predetermined period of time at a first predetermined temperature; (c) exposing the second population to an equal amount of solvent, which was used to dissolve the test compound, and a subinducing amount of a known cancer chemotherapeutic agent for said first predetermined period of time at said first predetermined temperature; (d) adding a reporter
  • side-by-side screening can be modified to accommodate the above described screening methodologies which utilize microscopic observations of changes in cellular morphology, cell cycle or observations of cellular culture growth rate. Because these modified assays do not follow caspase cascade activation, they do not require addition of a reporter compound.
  • the assays described in this section can also be used to screen for compositions that are selective for cell or tissue type. Such methodologies comprise side-by-side comparisons screening the affect of a given test compound on one cell or tissue type as compared to other cell or tissue types. In such an embodiment, cultures of each of the compared cell or tissue types comprise cells having elevated levels of expression of CCT.
  • the invention also relates to a method for assaying the cell or tissue selectivity of a potentially therapeutically effective antineoplastic compound that functions as an activator of the caspase cascade in viable cultured eukaryotic cells having an intact cell membrane and expressing elevated levels of CCT comprising: (a) obtaining a first population of viable cultured eukaryotic cells, each of which has an intact cell membrane and each of which expresses elevated levels of the CCT, by culturing said eukaryotic cells in a cell growth medium under conditions which result in growth; (b) obtaining a second population of viable cultured eukaryotic cells, each of which having an intact cell membrane and expressing elevated levels of the CCT by culturing said eukaryotic cells in a cell growth medium under conditions which result in growth; (c) separately exposing the first and second populations to a predetermined amount of a test compound for a predetermined period of time at a predetermined temperature; (d) adding to said first and second populations a reporter compound having at
  • the first population of cells can express a cancer phenotype that is not expressed in the second population of cells.
  • this method may be used to identify compounds that while specific for cancerous cells, do not affect non-cancerous cells.
  • side-by-side screening can be modified to accommodate the above described screening methodologies which utilize microscopic observations of changes in cellular morphology, cell cycle or observations of changes in cellular culture growth rate. Because these modified assays do not follow caspase cascade activation, they do not require addition of a reporter compound.
  • the invention further relates to a method to further determine the specificity of anticancer agents by determining the ability of the agent to arrest the cell cycle during a particular phase prior to apoptosis.
  • a time course of test compound treatment determines the phase of the cell cycle arrest that precedes apoptosis.
  • the G2M, S/G2M and Gl phases are the major phases in the cell cycle when one cell divides to become two daughter cells.
  • the cycle starts from a resting quiescent cell (GO phase) which is stimulated by growth factors leading to a decision (Gl phase) to replicate its DNA. Once the decision is made, the cell starts replicating its DNA (S-phase) and then into a G2 phase before finally dividing into two daughter cells.
  • S-phase resting quiescent cell
  • S-phase replicating its DNA
  • G2 phase a G2 phase
  • Cells which then undergo apoptosis contain fragmented DNA in amounts that are less that in the Gl phase and hence are called sub-Gl.
  • a compound leading to a Gl or G2M or S phase arrest and no apoptosis at 24 hr treatment, and leading to apoptosis at 48 hr treatment as determined by the presence of a sub-Gl peak indicates that the test compound arrests the cell cycle at the respective stage before inducing apoptosis. See Sherr, C. J., Cancer Res. 60:3689-3695 (2000), for a discussion of cancer cell cycles.
  • the invention relates to determining the specificity of a test compound by determining at what phase the cell cycle is arrested by the test compound prior to apoptosis.
  • Determining the specificity of a test compound to arrest the cell cycle during a particular phase prior to apoptosis comprises (a) obtaining at least one population of viable cultured cancer cells having intact cell membranes which have expression of CCT from a cell growth medium under conditions conducive to growth; (b) combining the at least one population with a predetermined amount of at least one test compound dissolved in a solvent for a predetermined period of time at a predetermined temperature thereby generating a first volume; and (c) determining at what phase the cell cycle is arrested.
  • the cells are incubated with a range of concentrations of test compound (e.g. 0.02 ⁇ M to 5 ⁇ M) for 6 h under normal growth conditions and control cultures are treated with DMSO vehicle. The cells are then treated e.g. for 20 min with 800 nM Syto 16. Cytospin preparations are then prepared and the samples are viewed by fluorescent microscopy using a fluorescein filter set. For each concentration of test compound, the number of mitotic figures are counted and expressed as a percentage of the total number of cells. Three fields from each condition are evaluated and the mean and SEM is calculated and plotted as a function of drug concentration. Another method is to simply stain the nuclei with Propidium Iodide and analyze the DNA content using a Fluorescence Activated Cell Sorter and Cell Quest Software (Becton Dickinson).
  • test compound e.g. 0.02 ⁇ M to 5 ⁇ M
  • Reporter compounds may be used as a means for measuring caspase cascade activity in the whole-cell assays of the present invention.
  • Typical reporter compounds include fluorogenic, chromogenic or chemiluminescent compounds applied to cells or tissues containing cells at a concentration of about 0.01 nanomolar to about 0.1 molar, or an equivalent amount of a salt or prodrug thereof. A concentration of about 10 micromolar may be used.
  • the test compounds may be presented to the cells or cell lines dissolved in a solvent.
  • solvents include, DMSO, water and/or buffers.
  • DMSO may be used in an amount below 2%.
  • DMSO may be used in an amount of 1% or below.
  • DMSO functions as a solubilizer for the test compounds and not as a permeabilization agent.
  • the amount of solvent tolerated by the cells must be checked initially by measuring cell viability or caspase induction with the different amounts of solvent alone to ensure that the amount of solvent has no effect on the cellular properties being measured.
  • Suitable buffers include cellular growth media, for example Iscove's media (Invitrogen Corporation) with or without 10% fetal bovine serum.
  • Other known cellular incubation buffers include phosphate, PIPES or HEPES buffers.
  • the cells can be derived from any organ or organ system for which it is desirable to find a potentially therapeutically effective antineoplastic compound that functions as an activator of the caspase cascade in viable cultured eukaryotic cells having an intact cell membrane.
  • Cellular genotypes for screening of test compounds include, but are not limited to, cells that are P53 negative, Bcl-2 over expressing, Bcl-xL over expressing, ataxia telengiectasia mutated (e.g. ATCC CRL 7201), multi-drug resistance (e.g.
  • P-glycoprotein over expressing ATCC CRL-1977
  • DNA mismatch repair deficiency e.g., defects in hMSH2, hMSH3, hMSH ⁇ , hPMS2, or hPMSl
  • HL-60 cells ATCC CCL-240
  • SH-SY5Y cells ATCC CRL-2266
  • Jurkat cells ATCC TIB- 152
  • surviving over expressing e.g. ATCC CCL- 185
  • bcr/abl mutated eg ATCC CCL-243
  • pi 6 mutated e.g. ATCC CRL-2336
  • Brca2 mutated e.g. ATCC CRL-2336
  • Suitable solubilizers may be used for presenting reporter compounds to cells or cell lines.
  • Solubilizers include aqueous solutions of the test compounds in water-soluble form, for example as water-soluble salts.
  • the test compounds may be dissolved in a buffer solution containing 20% sucrose (Sigma) 20 mM DTT (Sigma), 200 mM NaCl (Sigma), and 40 mM Na PIPES buffer pH 7.2 (Sigma).
  • Permeabilization agents include, but are not limited to, NP-40, n-octyl-O-D-glucopyranoside, n-octyl-O-D- thioglucopyranoside, taurocholic acid, digitonin, CHAPS, lysolecithin, dimethyldecylphosphine oxide (APO-10), dimethyldodecylphosphine oxide (APO- 12), N,N-bis-(3-D-gluconamidopropyl)cholamide (Big Chap), N,N-bis-(3-D-gluconamidopropyl)deoxycholamide (Big Chap, deoxy), BRIG-35, hexaethyleneglycol (C10E6), C10E8, C12E6, C12
  • the cell lines are exposed to a predetermined amount of test compounds at concentrations in the range from about 1 picomolar to about 1 millimolar, or about 1-10 micromolar.
  • the predetermined period of time may be about 1 minute to less than about 24 hours, or 1-24 hours, or 3, 5, or 24 hours.
  • the predetermined temperature may be about 4 0 C to about 50 0 C, or about 37 0 C.
  • REU Relative Fluorescence Unit values
  • the potency of caspase cascade activation is determined by the ratio of the Net RFU value for a test compound to that of control samples as follows:
  • Preferred test compounds are those indicating a ratio of 2 or greater and most preferably with a measured ratio greater than a statistically significant value calculated as (Ave Control RFU + 4 x SDcontr o i) / (Ave Control RFU) for that run.
  • Examples of high throughput instrumentation which can be used according to the present invention are well known in the art.
  • Non-limiting examples of such instruments include ImageTrak® (Packard BioScience), the FLIPR® system, Spectramax Gemini or FMax (Molecular Devices Corporation, Sunnyvale, CA), VIPRTM II Reader (Aurora Biosciences Corporation, San Diego, Ca), Fluoroskan II (GMI, Inc., Albertville, MN), Fluoroskan Ascent (Labsystems, Franklin, MA), Cytofluor or Cytofluor 4000 (Perkin Elmer Instruments), Cytofluor 2300 (Millipore, FLx800TBID, FLx800TBIDE, ELx808, ELx800, FL600 (Bio-Tek Instruments), Spectrafluora, Spectrofluora Plus, Ultra or Polarion (Tecan AG), MFX (Dynex Technologies, Chantilly, VA), Fluoro Count (Packard Instruments Co.), NOVOstar, POLARstar Galaxy or FLU
  • this method can be carried out by comparing the members of the chemical library with the crystal structure of the CCT using computer programs known to those of skill in the art (e.g., Dock, Kuntz, I. D. et al, Science, 257:1078-1082 (1992); Kuntz, I. D. et al, J. MoI. Biol, 161:269 (1982); Meng, E. C, et al, J. Comp. Chem., 13: 505-524 (1992) or CAVEAT).
  • computer programs known to those of skill in the art e.g., Dock, Kuntz, I. D. et al, Science, 257:1078-1082 (1992); Kuntz, I. D. et al, J. MoI. Biol, 161:269 (1982); Meng, E. C, et al, J. Comp. Chem., 13: 505-524 (1992) or CAVEAT).
  • the library of molecules to be searched can be any library, such as a database (i.e., online, offline, internal, external) which comprises crystal structures, coordinates, chemical configurations or structures of molecules, compounds or drugs to be assessed or screened for their ability to bind to CCT.
  • a database i.e., online, offline, internal, external
  • databases for drug design such as the Cambridge Structural Database (CSD), which includes about 100,000 molecules whose crystal structures have been determined or the Fine Chemical Director (FCD) distributed by Molecular Design Limited (San Leandro, Calif.) can be used. See Allen, F. H., et al, Acta Crystallogr. Section B, 55:2331 (1979).
  • a library such as a database, biased to include an increased number of members which comprise indole rings, hydrophobic moieties and/or negatively-charged molecules can be used.
  • a drug or molecule which binds or fits into a cavity or pocket on the surface of CCT can be used alone or in combination with other drugs (as part of a drug cocktail) to prevent, ameliorate or treat conditions responsive to induction of apoptosis.
  • a drug designed or formed by a method described herein is also the subject of this invention.
  • Iodine monochloride (380 mg, 2.3 mmol) was added to 4-(3-(2- methoxyphenyl)-7 ⁇ -[l,2,4]triazolo[3,4-b][l,3,4]thiadiazin-6-yl)benzenamine (153 mg, 0.45 mmol) dissolved in glacial acetic acid at 10 0 C. The mixture was stirred at the same temperature for 0.5h, more iodine monochloride (250 mg, 1.5 mmol) was added and stirred for 2h at room temperature.
  • reaction mixture was evaporated to dryness and dissolved in 50 mL of ethyl acetate, washed with saturated NaCO 3 , water, 10% Na 2 S 2 O 3 , water and saturated NaCl. The organic layer was dried over anhydrous Na 2 SO 4 , filtered and concentrated. The residue was purified by chromatography (40% acetone/hexane) to give title compound (47 mg, 0.080 mmol, 18%).
  • the T-labeled azido compound was prepared by a procedure similar as the non-labeled compound by using 6-(4-amino-3,5-ditritiumphenyl)-3-(2- methoxyphenyl)-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazine as the starting materials .
  • [l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazine was prepared by reaction of 6-(4- amino-3,5-dibromophenyl)-3-(2-methoxyphenyl)-7H-[l,2,4]triazolo[3,4- Z>][l,3,4]thiadiazine with T 2 in the presence of a metal catalyst.
  • the T-labeled azido compound was purified by ⁇ PLC, with chemical and radiochemical purity of >98%, and specific activity of 40-50 Ci/mmol.
  • Namalwa cells were grown in RPMI 1640 media containing 25 mM ⁇ epes and L-glutamine (Gibco) supplemented with 10 % FCS and penicillin/streptomycin and harvested by centrifugation (200 x g, 5 minutes). To determine electrophoretic mobility of compound-bound proteins, 1 x 107 Namalwa cells were resuspended in 1 mL RPMI 1640 media with 10 % FCS.
  • An untreated, unlabelled Namalwa lysate was prepared in parallel by harvesting 1 x 107 Namalwa cells by centrifugation (200 x g, 5 minutes), washed one time with 1 mL phosphate buffered saline (PBS) and fractionated into cytoplasmic and nuclear fractions using the NE-PER kit (Pierce). This cytoplasmic fraction will be referred to as "cold Namalwa lysate.” These lysates were then concentrated with an Ultracel YM30 concentrator (Centricon) to a final volume of 0.04 ml.
  • a volume of 0.01 ml of either treated or cold concentrated Namalwa lysate was resolved by running the sample on a p ⁇ 3 - 10 nonlinear (NL) isoelectric focusing strip (Invitrogen Corporation) at 500 V for 4 hours. Strips were then equilibrated first in IX sample buffer with sample reducing agent (Invitrogen), and then in IX sample buffer with iodoacetimide. Equilibrated strips were then resolved in the second dimension by SDS-PAGE at 150 V for 1.5 hours.
  • NL nonlinear
  • Both gels (cold Namalwa lysate or treated Namalwa lysate) were then stained with 1 % Coomassie Brilliant Blue in 40% methanol, 7.5% acetic acid for one hour, then destained overnight with 40 % methanol, 7.5 % acetic acid.
  • the gel with the treated Namalwa sample was then treated with Amplify (Amersham Biosciences, Piscataway, NJ), dried, and exposed to film at -80 0 C in order to determine the electrophoretic position of compound- bound proteins. This film was then used to isolate gel samples from the gel with the cold Namalwa lysate for subsequent tryptic digestion and LC/MS/MS analysis. Trypsin digestion:
  • Namalwa cells were grown in RPMI 1640 media containing 25 mM Hepes and L-glutamine (Gibco) supplemented with 10 % FCS and penicillin/streptomycin and harvested by centrifugation (200 x g, 5 minutes). Cells were then treated with 0.2 ⁇ M of 6-(4-azido-3,5-ditritiumphenyl)-3-(2- methoxyphenyl)-7H-[l,2,4]triazolo[3,4-6][l,3,4]thiadiazine (Example 3) in DMSO for 1 hour at 37 0 C and then UV crosslinked for 10 minutes.
  • lysed cells were washed one time with 1 mL phosphate buffered saline (PBS) and lysed in 0.1 mL RIPA buffer (10x RIPA supplied by Upstate) and 0.1% Protease Inhibitor Cocktail (Sigma). The lysed cells were spun at 20,000xg for 10 min and the supernatant collected and are referred to as "treated Namalwa lysate.”
  • PBS phosphate buffered saline
  • RIPA buffer 10x RIPA supplied by Upstate
  • Protease Inhibitor Cocktail Sigma
  • Immunoprecipitations were set up with 50 ⁇ L treated Namalwa lysate and 2 ⁇ g antibody for CCTl (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), CCT2 (Abeam, Cambridge, MA), CCT3 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), CCT4 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), CCT5 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), CCT6 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), CCT7 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), CCT8 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), or Nmi (Santa Cruz Biotechnology, Inc, Santa Cruz, CA).
  • Antibodies were incubated with treated Namalwa lysate overnight. Lysates and antibody were then added to 50 ul pre- washed Protein G sepharose beads (Upstate/Millipore). Sepharose was washed three times with RIPA. Sample buffer with DTT was added to beads, heated, and subjected to SDS-PAGE. The SDS-PAGE gel was then Coomassie stained, treated with Amplify (Amersham Biosciences, Piscataway, NJ), dried, and exposed to film to visualize compound-bound proteins.
  • CCT4 protein was incubated with either DMSO, 3-(3- methoxyphenyl)-6-(4-nitrophenyl)-7H-[ 1 ,2,4]triazolo[3,4-6][ 1 ,3,4]thiadiazine inactive analog (compound 1), or 6-(3-amino-4-methylphenyl)-3-(2- methoxyphenyl)-7H-[ 1 ,2,4]triazolo[3,4- ⁇ ][ 1 ,3,4]thiadiazine active analog (compound 2) for 1 hour at room temperature prior to addition of 6-(4-azido- 3,5-ditritiumphenyl)-3-(2-methoxyphenyl)-7H-[l,2,4]triazolo[3,4- 6][1, 3,4]thiadiazine.
  • the proteins were exposured to UV for 10 minutes at 3.5 cm distance and then subjected to SDS-PAGE.
  • the SDS-PAGE gel was then Coomassie stained, treated with Amplify (Amersham Biosciences, Piscataway, NJ), dried, and exposed to film to visualize compound-bound proteins.
  • purified proteins were subjected to SDS-PAGE and stained with 1 % Coomassie Brilliant Blue in 40% methanol, 7.5% acetic acid for one hour, then destained overnight with 40 % methanol, 7.5 % acetic acid.
  • This binding can be competed with unlabelled active analog 6-(3-amino-4-methylphenyl)-3-(2- methoxyphenyl)-7H-[l,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazine (compound 2), but not by inactive analog 3-(3-methoxyphenyl)-6-(4-nitrophenyl)-7H- [l,2,4]triazolo[3,4-6][l,3,4]thiadiazine (compound 1).
  • PLKl full length cDNA was in vitro translated using TNT T7 Coupled Reticulocyte Lysate System (Promega, Madison, WI) with either DMSO or 1 ⁇ M 3-(3-methoxyphenyl)-6-(4-nitrophenyl)-7H-[l ,2,4]triazolo[3,4- ⁇ ][l,3,4]thiadiazine inactive analog (compound 1) or 6-(3-amino-4- methylphenyl)-3-(2-methoxyphenyl)-7H-[l,2,4]triazolo[3,4- 6][l,3,4]thiadiazine active analog (compound 2) included in the translation reaction at 30 0 C for 90 minutes.
  • TNT T7 Coupled Reticulocyte Lysate System Promega, Madison, WI
  • Translated PLKl protein was immunoprecipitated from translation reactions by adding 200 ⁇ L IX RIPA buffer and 2 ug PLKl mouse antibody (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) overnight.
  • DMSO or 6-(3-amino-4-methylphenyl)-3-(2- methoxyphenyl)-7H-[l,2,4]triazolo[3,4-£][l,3,4]thiadiazine active analog (compound 2) were added post-translation to the immunoprecipitation of a subset of samples treated with DMSO, in order to control for effects of the compound on PLKl kinase activity as opposed to folding.
  • Sepharose beads were then resuspended in 50 ⁇ L IVK buffer, 20 ⁇ L of which was then used for an in vitro kinase assay by adding 5 ⁇ g purified casein protein as a substrate, 25 ⁇ M ATP and 10 ⁇ Ci ⁇ - 32 P-ATP (Perkin Elmer) per reaction. Reactions were incubated at 30 0 C for 30 minutes, then subjected to SDS-PAGE. Gels were dried and exposed to film to visualize 32 P kinase activity. In parallel, 5 ⁇ L of the original immunoprecipitation sample was subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted for PLKl protein with a rabbit primary antibody.
  • Figure 3 showed that PLKl folding is significantly inhibited by active analog 6-(3-amino-4- methylphenyl)-3-(2-methoxyphenyl)-7H-[ 1 ,2,4]triazolo[3,4- 6][l,3,4]thiadiazine (compound 2), resulting in decreased kinase activity (fig 3b, c), but not a decrease in total PLKl protein synthesis (fig 3a).

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  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Genetics & Genomics (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un procédé de traitement, prévention ou amélioration d’une maladie sensible à l’induction de la cascade de la caspase chez un animal, comprenant l’administration à l’animal d’un composé qui se lie à CCT. La présente invention concerne en outre des procédés de criblage utiles pour la découverte de médicament de composés induisant l’apoptose. En particulier, la méthodologie de criblage concerne l’utilisation de CCT en tant que cible pour la découverte d'activateurs d’apoptose utiles en tant qu’agents anticancéreux. Les procédés de criblage de la présente invention peuvent utiliser des essais de liaison homogènes ou hétérogènes utilisant CCT purifié ou partiellement purifié ; ou des essais sur des cellules entières utilisant des cellules ayant des taux altérés de CCT. L’invention concerne en outre l’utilisation de la 6-aryl-3-aryl-7H-[1,2,4]triazolo[3,4-a][1,3,4]thiadiazine ou d’un dérivé qui se lie à CCT et peut, en conséquence, être utilisée pour induire des anticorps utiles pour la découverte de médicament. En variante, de la 6-aryl-3-aryl-7H-[1,2,4]triazolo[3,4-a][1,3,4]thiadiazine marquée ou un dérivé est utilisé pour des essais de liaison compétitive pour la découverte de médicament. De tels essais permettent le criblage à rendement élevé de bibliothèques chimiques pour des activateurs d’apoptose.
PCT/US2009/004106 2008-07-16 2009-07-16 Composés et procédés d’induction de l’apoptose par interaction avec la chaperonine cct WO2010008558A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019166651A1 (fr) * 2018-03-01 2019-09-06 Pro Test Diagnostics Ab Procédés de détection de dopage sanguin autologue

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008011045A2 (fr) * 2006-07-18 2008-01-24 Cytovia, Inc. 3-ARYL-6-ARYL-7H-[1,2,4]TRIAZOLO[3,4-b][1,3,4]THIADIAZINES ET ANALOGUES CONSTITUANT DES ACTIVATEURS DE CASPASES ET DES INDUCTEURS D'APOPTOSE ET UTILISATION DE CES COMPOSÉS

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008011045A2 (fr) * 2006-07-18 2008-01-24 Cytovia, Inc. 3-ARYL-6-ARYL-7H-[1,2,4]TRIAZOLO[3,4-b][1,3,4]THIADIAZINES ET ANALOGUES CONSTITUANT DES ACTIVATEURS DE CASPASES ET DES INDUCTEURS D'APOPTOSE ET UTILISATION DE CES COMPOSÉS

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GRANTHAM, J. ET AL.: "Substantial CCT activity is required for cell cycle progression and cytoskeletal organization in mammalian cells", EXPERIMENTAL CELL RESEARCH, vol. 312, no. 12, 2006, pages 2309 - 2324 *
LIU, XIAOQI ET AL.: "CCT Chaperonin Complex Is Required for the Biogenesis of Functional Plkl", MOLECULAR AND CELLULAR BIOLOGY, vol. 25, no. 12, 2005, pages 4993 - 5010 *
RAMOS, B. ET AL.: "Inhibition of CTP:Phosphocholine Cytidyltransferase by C2- Ceramide and Its Relationship to Apoptosis", MOLECULAR PHARMACOLOGY, vol. 62, no. 5, 2002, pages 1068 - 1075 *
WIEDER, T.: "Induction of Ceramide-mediated Apoptosis by the Anticancer Phospholipid Analog, Hexadecylphosphocholine", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 18, 1998, pages 11025 - 11031 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019166651A1 (fr) * 2018-03-01 2019-09-06 Pro Test Diagnostics Ab Procédés de détection de dopage sanguin autologue

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