• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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    • 专利摘要:
    Uses and applications derived from the discovery of a novel binding site of ΙΚΚ-β, such as method of screening a therapeutic agent as drug candidate for treating cancer, inflammation, or other diseases/disorders, are provided.
    公开号:AU2013217247A1
    申请号:U2013217247
    申请日:2013-01-31
    公开日:2013-10-03
    发明作者:Zhihong Jiang；Ting Li；Liang Liu；Kam Wai Wong；Hua Zhou
    申请人:Macau Univ of Science and Technology；
    IPC主号:C12Q1-68
    专利说明:
    WO 20131118033 PCT/IB2013/050812 NOVEL BINDING SrrE OF IKK-P FIELD OF INVENTION 5 [0001] This invention relates to a novel binding site of IKK-p, and in particular uses and applications derived from the discovery of this novel binding site. BACKGROUND OF INVENTION [0002] Diseases such as cancers and inflammations are deliberating and may be fatal. 10 Thus, it is essential to develop methods for effectively screening therapeutic agents as drug candidates for treating these diseases. It is also important to develop reliable methods for screening and/or diagnosing patients having these disorders so that they can receive appropriate treatment as early as possible. 15 SUMMARY OF INVENTION [0003] In the light of the foregoing background, it is an object of the present invention to provide a new method for screening therapeutic agents as drug candidates for treating these diseases. 20 [0004] Accordingly, the present invention, in one aspect, is a method of screening a therapeutic agent as a drug candidate for treating cancer, inflammation, neurodegenerative disease, immunological disorder, or arthritic disorder comprising: [0005] a) exposing said agent to an assay comprising IKK-p; [0006] b) detecting whether said agent binds to cysteine-46 (Cys-46 or C46) residue of 25 IKK-; [0007] c) detecting whether said agent inhibits kinase activity of IKK-p upon said binding in step (b) ; and 1 WO 2013/118033 PCT/IB2013/050812 [0008] d) identifying a drug candidate that performs said binding action of step (b) and said inhibition action of step (c). 30 [00091 In an exemplary embodiment of the present invention, at least one binding site of IKK-p is mutated. In a further exemplary embodiment, said mutated binding site is selected from a group consisting of phenylalanine residue, serine-177/181 residue, allosteric binding site of 1KK-@, and cysteine residue except cysteine-46 residue. [0010] In an even further exemplary embodiment, said mutated cysteine or phenylalanine 35 residue is selected from a group consisting of cysteine-12 (Cys-12 or C12) residue, phenylalanine-26 (Phe-26 or F26, alternatively known as ATP binding site) residue, cysteine-59 (Cys-59 or C59) residue, cysteine-99 (Cys-99 or C99) residue, cysteine-1 14 (Cys-114 or C1 14) residue, cysteine-1 15 (Cys-115 or Cl 15) residue, cysteine-179 (Cys 179 or C179) residue, cysteine-215 (Cys-215 or C215) residue, cysteine-299 (Cys-299 or 40 C299) residue, cysteine-370 (Cys-370 or C370) residue, cysteine-412 (Cys-412 or C412) residue, cysteine-444 (Cys-444 or C444) residue, cysteine-464 (Cys-464 or C464) residue, cysteine-524 (Cys-524 or C524) residue, cysteine-618 (Cys-618 or C618) residue, cysteine-662/716 (Cys-662/716 or C6621716) residue, cysteine-751 (Cys-751 or C751) residue; said mutation is a point mutation from cysteine or phenylalanine to alanine. 45 [0011] In another embodiment, said cancer is selected from a group consisting of lung cancer, colon cancer, liver cancer, breast cancer, prostate cancer, cervical cancer, acute promyelocytic leukemia (APL), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma, Hodgkin's disease, chronic lymphocytic leukemia (CLL), myelodysplastic syndrome, 50 Adult T-cell leukemia (ATL), Burkitt's lymphoma, B-cell lymphoma, primary malignant lymphocytes, B-cell chronic lymphocytic leukemia (B-CLL), human THP-1 leukemia and multiple myeloma. In yet another embodiment, said inflammation is selected from a group consisting of ear edema, dermatitis, ear inflammation, and arthritis. [0012] In another exemplary embodiment, said neurodegenerative disease is selected 55 from a group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral 2 WO 2013/118033 PCT/IB2013/050812 sclerosis, ataxia telangiectasia, spinocerebellar atrophy, multiple sclerosis, and Huntington's chore a. [0013] In yet another exemplary embodiment, said immunological disorder is selected from a group consisting of allergic rhinitis, allergic dermatitis, allergic contact dermatitis, 60 allergic shock, asthma, papular urticaria, leucoderma, hypersensitivity vasculitis, hypersensitivity pneumonia, ulcerative colitis, glomerulonephritis, drug rashes, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, multiple sclerosis, hyperthyroidism, idiopathic thrombocytopenic, autoimmune hemolytic anemia, allograft rejection, and hemolytic transfusion reaction. 65 [0014] In a further exemplary embodiment, said arthritic disorder is selected from a group consisting of rheumatoid arthritis, ankylosing spondylitis, gout, periarthritis, osteoarthritis, Reiter syndrome, psoriatic arthritis, post-traumatic arthritis, and enteropathic arthritis. [0015] According to another aspect of the present invention, a method for diagnosing 70 cancer, inflammation, neurodegenerative disease, immunological disorder, or arthritic disorder in a patient is provided, comprising: [0016] a) obtaining a sample from said patient; (0017] b) contacting said sample with a compound that binds to cysteine-46 residue of IKK-p of said sample; 75 [0018] c) detecting binding of said compound to IKK-p in said sample; [0019] d) detecting inhibition action on kinase activity of IKK-P by said compound upon said binding in step (c); and (0020] e) diagnosing said patient as having a likelihood to develop cancer, inflammation, neurodegenerative disease, immunological disorder, or arthritic disorder if said 80 compound cannot perform said binding action of step (c) and/or said inhibition action of step (d). 3 WO 2013/118033 PCT/IB2013/050812 [0021] In an exemplary embodiment of the present invention, at least one binding site of IKK-s is mutated. In a further exemplary embodiment, said mutated binding site is selected from a group consisting of, phenylalanine residue, serine-177/181 residue, 85 allosteric binding site, and cysteine residue except cysteine-46 residue. [0022] In an even further exemplary embodiment, said mutated cysteine or phenylalanine residue is selected from a group consisting of cysteine-12 residue, phenylalanine-26 (Phe 26 or F26, alternatively known as ATP binding site) residue, cysteine-59 residue, cysteine-99 residue, cysteine-114 residue, cysteine-115 residue, cysteine-179 residue, 90 cysteine-215 residue, cysteine-299 residue, cysteine-370 residue, cysteine-412 residue, cysteine-444 residue, cysteine-464 residue, cysteine-524 residue, cysteine-618 residue, cysteine-662/716 residue, and cysteine-751 residue; said mutation is a point mutation from cysteine or phenylalanine to alanine. [0023] In another embodiment, said cancer is selected from a group consisting of lung 95 cancer, colon cancer, and liver cancer, breast cancer, prostate cancer, cervical cancer, acute promyelocytic leukemia (APL), acute mycloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma, Hodgkin's disease, chronic lymphocytic leukemia (CLL), myelodysplastic syndrome, Adult T-cell leukemia (ATL), Burkitt's lymphoma, B-cell lymphoma, primary malignant 100 lymphocytes, B-cell chronic lymphocytic leukemia (B-CLL), human THP-1 leukemia, and multiple myeloma. In yet another embodiment, said inflammation is selected from a group consisting of ear edema, dennatitis, ear inflammation, and arthritis. [0024] In another exemplary embodiment, said neurodegenerative disease is selected from a group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral 105 sclerosis, ataxia telangiectasia, spinocerebellar atrophy, multiple sclerosis, and Huntington's chorea. [0025] In yet another exemplary embodiment, said immunological disorder is selected from a group consisting of allergic rhinitis, allergic dermatitis, allergic contact dermatitis, allergic shock, asthma, papular urticaria, leucoderma, hypersensitivity vasculitis, 110 hypersensitivity pneumonia, ulcerative colitis, glomerulonephritis, drug rashes, systemic 4 WO 2013/118033 PCT/IB2013/050812 lupus erythematosus, rheumatoid arthritis, scleroderma, multiple sclerosis, hyperthyroidism, idiopathic thrombocytopenic, autoimmune hemolytic anemia, allograft rejection, and hemolytic transfusion reaction. [0026] In a further exemplary embodiment, said arthritic disorder is selected from a 115 group consisting of rheumatoid arthritis, ankylosing spondylitis, gout, periarthritis, osteoarthritis, Reiter syndrome, psoriatic arthritis, post-traumatic arthritis, and enteropathic arthritis. [0027] In a further aspect of the present invention, a method of screening a patient to have a likelihood to develop cancer, inflammation, neurodegenerative disease, 120 immunological disorder, or arthritic disorder is provided, comprising: [0028] a) obtaining a sample from said patient; [0029] b) contacting said sample with a compound that binds to cysteine-46 residue of IKK-p of said sample; [0030] c) detecting binding of said compound to IKK-p in said sample; 125 [0031] d) detecting inhibition action on kinase activity of IKK-p by said compound upon said binding in step (d); and [0032] e) identifying said patient as having a likelihood to develop cancer, inflammation, neurodegenerative disease, immunological disorder, or arthritic disorder if said compound cannot perform said binding action of step (c) and/or said inhibition action of 130 step (d). [0033] In an exemplary embodiment of the present invention, at least one binding site of IKK-p is mutated. In a further exemplary embodiment, said mutated binding site is selected from a group consisting of phenylalanine residue, serine-177/181 residue, allosteric binding site of IKK-p, and cysteine residue except cysteine-46 residue. In an 135 even further exemplary embodiment, said mutated cysteine or phenylalanine residue is selected from a group consisting of cysteine-12 residue, phenylalanine-26 (Phe-26 or F26, alternatively known as ATP binding site) residue, cysteine-59 residue, cysteine-99 5 WO 2013/118033 PCT/IB2013/050812 residue, cysteine-114 residue, cysteine-115 residue, cysteine-179 residue, cysteine-215 residue, cysteine-299 residue, cysteine-370 residue, cysteine-412 residue, cysteine-444 140 residue, cysteine-464 residue, cysteine-524 residue, cysteine-618, cysteine-662/716 residue, and cysteine-751 residue; said mutation is a point mutation from cysteine or phenylalanine to alanine. [0034] In another embodiment, said cancer is selected from a group consisting of lung cancer, colon cancer, liver cancer, breast cancer, prostate cancer, cervical cancer, acute 145 promyelocytic leukemia (APL), acute mycloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma, Hodgkin's disease, chronic lymphocytic leukemia (CLL), myclodysplastic syndrome, Adult T-cell leukemia (ATL), Burkitt's lymphoma, B-cell lymphoma, primary malignant lymphocytes, B-cell chronic lymphocytic leukemia (B-CLL), human THP-1 leukemia 150 and, multiple myeloma. In yet another embodiment, said inflammation is selected from a group consisting of ear edema, dermatitis, ear inflammation, and arthritis. [0035] In another exemplary embodiment, said neurodegenerative disease is selected from a group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, spinocerebellar atrophy, multiple sclerosis, and 155 Huntington's chorea. [0036] In yet another exemplary embodiment, said immunological disorder is selected from a group consisting of allergic rhinitis, allergic dermatitis, allergic contact dermatitis, allergic shock, asthma, papular urticaria, leucoderma, hypersensitivity vasculitis, hypersensitivity pneumonia, ulcerative colitis, glomerulonephritis, drug rashes, systemic 160 lupus erythematosus, rheumatoid arthritis, scleroderma, multiple sclerosis, hyperthyroidism, idiopathic thrombocytopenic, autoimmune hemolytic anemia, allograft rejection, and hemolytic transfusion reaction. [0037] In a further exemplary embodiment, said arthritic disorder is selected from a group consisting of rheumatoid arthritis, ankylosing spondylitis, gout, periarthritis, 165 osteoarthritis, Reiter syndrome, psoriatic arthritis, post-traumatic arthritis, and enteropathic arthritis. 6 WO 2013/118033 PCT/IB2013/050812 [0038] In yet a further aspect of the present invention, a method for treating cancer, inflammation, neurodegenerative disease, immunological disorder, or arthritic disorder is provided, comprising administering an effective amount of a therapeutic agent to a 170 patient in need thereof, wherein said patient harbors gene mutations on at least one binding site of IKK-p; said mutated binding site is selected from a group consisting of phenylalanine residue, serine-177/181 residue, allosteric binding site of IKK-P, and cysteine residue except cysteine-46 residue. [0039] In an exemplary embodiment, said mutated cysteine or phenylalanine residue is 175 selected from a group consisting of cysteine-12 residue, phenylalanine-26 (Phe-26 or F26, alternatively known as ATP binding site) residue, cysteine-59 residue, cysteme-99 residue, cysteine-114 residue, cysteine-115 residue, cysteine-179 residue, cysteine-215 residue, cysteine-299 residue, cysteine-370 residue, cysteine-412 residue, cysteine-444 residue, cysteine-464 residue, cysteine-524 residue, cysteine 618 residue, cysteine 180 662/716 residue, and cysteine-751 residue; said mutation is a point mutation from cysteine or phenylalanine to alanine. In another embodiment, said therapeutic agent binds to cysteine-46 residue of IKK-p and inhibits the kinase activity of IKK-p upon said binding. [0040] In another embodiment, said cancer is selected from a group consisting of lung 185 cancer, colon cancer, liver cancer, breast cancer, prostate cancer, cervical cancer, acute promyelocytic leukemia (APL), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma, Hodgkin's disease, chronic lymphocytic leukemia (CLL), myelodysplastic syndrome, Adult T-cell leukemia (ATL), Burkitt's lymphoma, B-cell lymphoma, primary malignant 190 lymphocytes, B-cell chronic lymphocytic leukemia (B-CLL), human THP-1 leukemia and multiple myeloma. In yet another embodiment, said inflammation is selected from a group consisting of ear edema, dermatitis, ear inflammation, and arthritis. [0041] In another exemplary embodiment, said neurodegenerative disease is selected from a group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral 7 WO 2013/118033 PCT/IB2013/050812 195 sclerosis, ataxia telangiectasia, spinocerebellar atrophy, multiple sclerosis, and Huntington's chorea. [0042] In yet another exemplary embodiment, said immunological disorder is selected from a group consisting of allergic rhinitis, allergic dermatitis, allergic contact dermatitis, allergic shock, asthma, papular urticaria, leucoderma, hypersensitivity vasculitis, 200 hypersensitivity pneumonia, ulcerative colitis, glomerulonephritis, drug rashes, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, multiple sclerosis, hyperthyroidism, idiopathic thrombocytopenic, autoimnimune hemolytic anemia, allograft rejection, and hemolytic transfusion reaction. [0043] In a further exemplary embodiment, said arthritic disorder is selected from a 205 group consisting of rheumatoid arthritis, ankylosing spondylitis, gout, periarthritis, osteoarthritis, Reiter syndrome, psoriatic arthritis, post-traumatic arthritis, and enteropathic arthritis. BRIEF DESCRIPTION OF FIGURES 210 [0044] Fig. I shows the amino acid sequence of IKK-P protein as shown in SEQ ID NO: tin which the mutated residues are underlined. [0045] Fig. 2A shows the synthesis of biotinylated DMY (DMY-biotin), whereas Figs. 2B to 2D show the comparison of synthesized DMY-biotin and DMY on T cell proliferation, NF-B activation, as well as IKK-$ kinase activity according to one 215 embodiment of the present invention. [0046] Fig. 3 shows the study of the binding site of DMY and DMY-biotin according to one embodiment of the present invention. [0047] Fig. 4 shows the study of a new drug binding site involved in IKK-P using IKK 3 displacement binding assay according to one embodiment of the present invention. 8 WO 2013/118033 PCT/IB2013/050812 220 [0048] Fig. 5 shows the study of DMY on its activity on dmug resistant phenotype of IKK-P mutants with cysteine-179 mutation (C179A) or ATP-binding site mutation (F26A) according to one embodiment of the present invention. [0049] Fig. 6 shows the study of DMY on its inhibition of the kinase activity of IKK mutants with cysteine-46 mutation (C46A), as well as form protein adduct with IKK-s 225 mutants (C46A) according to one embodiment of the present invention. [0050] Fig. 7 shows the study of DMY on its activity to suppress IKK-0 mutants with various single cysteine mutations according to one embodiment of the present invention. [0051] Fig. 8 shows the study of DMY on its ability to form protein adduct with IKK-P mutants with various single cysteine mutations according to one embodiment of the 230 present invention. (0052] Fig. 9 shows the study of DMY on its ability to suppress IKK-s - NF-KB signaling of both wild-type and IKK-P mutants with cysteine-46 mutation (C46A) in IKK- 4- deficient MEFs according to one embodiment of the present invention. (0053] Figs. 10A to 10 show the study of DMY on its effect on ear edema induced by 235 dinitrofluorobenzene according to one embodiment of the present invention. [0054] Figs. 1 IA to 1ID show the study of DMY on its effect on arthritis model induced by collagen II according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 240 [0055] As used herein and in the claims, "comprising" means including the following elements but not excluding others. When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner. 9 WO 2013/118033 PCT/IB2013/050812 245 [0056] The present invention provides a new technical platform for identifying the action mechanisms of existing IKK-p inhibitors and screening of new IKK-$ inhibitors. In one exemplary embodiment, using the platform, dihydromyricetin (DMY) could directly suppress the kinase activity of IKK-p via a novel drug binding site, cysteine-46 (Cys-46) residue, rather than via known binding sites on IKK-$ such as ATP binding site, cysteine 250 179 (Cys-179) residue, serine-177/181 (Ser-177/181) residue, and allosteric binding site. In another exemplary embodiment, DMY could circumvent the drug resistance phenotype of IKK-p with mutations of Cys-179 residue or phenylalanine-26 (Phe-26 or F26, alternatively known as ATP binding site) residue. It could thus be deduced that the discovery of DMY with novel binding site of IKK- could be useful for patients 255 harboring gene mutation on IKK-$, especially on Cys-179 and ATP-binding regions (Phe-26 or F26). [0057] Since IKK-p plays a vital role in the regulation of NF-xr signaling pathway which in turn leads to the regulation of transcription of genes involved in important mechanisms within cells such as T-cell activation, the medicinal usages thereof have been 260 widely studied and published. For instance, IKK-p inhibitors have been proven to treat auto-immune diseases [Refs. 1-2], rheumatoid arthritis [Refs. 3-12], chronic obstructive pulmonary disease (COPD) and asthma [Refs. 11-27], cancer [Refs. 28-38], and diabetes [Refs. 39-42]. The references cited for each of the foregoing and hereinafter diseases in square bracket with "[Refs.xx]" with xx referring to the number of the corresponding 265 literatures on the "References" list. [0058] It can be deduced from the present invention that a compound or therapeutic agent that binds to cysteine-46 residue of IKK-P and inhibits the kinase activity of IKK-P can be used as inhibitors of IKK-p and NF-rcB. As such, it can be further deduced by one skilled in the art that the aforesaid compound or therapeutic agent can be used for the 270 treatment for the diseases described above as these diseases are associated with the activation of IKK-P and NF-4B. 10 WO 2013/118033 PCT/IB2013/050812 [00591 In addition, NF-rB activation could mediate the Abeta-associated phenotype in Alzheimer disease, suggesting the critical role of NF-cB activation in neurodegenerative diseases [Ref. 44] 275 [0060] It can also be deduced from the present invention that a compound or therapeutic agent that binds to cysteine-46 residue of IKK-p and inhibits the kinase activity of IKK-p can be used as suppressor of IKK-p/NF-icB activation. As such, it can be deduced by one skilled in the art that the aforesaid compound or therapeutic agent can be used for the treatment for the diseases described above as these diseases are associated with the 280 activation of NF-B signaling. [0061] Further, it can be deduced from the present invention that a compound or therapeutic agent that binds to cysteine-46 residue of IKK-p and inhibits the kinase activity of IKK-P can be used as suppressor of immune reaction and hypersensitivity. As such, it can be deduced by one skilled in the art that the aforesaid compound or 285 therapeutic agent can be used for the treatment for the diseases described above as these diseases are associated with the activation of immune reaction and hypersensitivity. [0062] It can be deduced from the present invention that a compound or therapeutic agent that binds to cysteine-46 residue of IKK-p and inhibits the kinase activity of IKK-p can be used as inhibitor of arthritis. As such, it can be deduced by one skilled in the art that 290 the aforesaid compound or therapeutic agent can be used for the treatment for the diseases described above as these diseases are associated with arthritis. [0063] The present invention is further defined by the following examples, which are not intended to limit the present invention. Reasonable variations, such as those understood by reasonable artisans, can be made without departing from the scope of the present 295 invention. [0064] Example 1 (0065] Site-directed Mutagenesis Assay 11 WO 2013/118033 PCT/IB2013/050812 [0066] This example describes the assays that the cysteine or phenylalanine residue was mutated to alanine or one by one to establish the technique platform. 300 [0067] Cloning and expression [0068] The FLAG-IKK-$ construct was used as a template to introduce the single point mutants having cysteine (C) residue or phenylalanine (F) residue replaced with alanine (A) including C12A, C46A, C59A, C99A, C114A, C115A, C215A, C299A, C370A, C412A, C444A, C464A, C524A, C618A, C751A and F26A mutations. These mutated 305 residues are underlined in the amino acid sequence (SEQ ID NO:1) as shown in Fig. 1. (0069] The site-directed mutagenesis was carried out using the Stratagene Quikchange Mutagenesis Kit accordingly to the manufacturer's instructions. The mutations of clones were confirmed by DNA sequencing. [0070] Example 2 310 [0071] Synthesis of Biotinylated DMY Assay, NF-wB Luciferase Reporter Assay, and IKK-P Kinase Assay [0072] This example describes the synthesis of biotinylated DMY and comparison of the actions of DMY and DMY-biotin on T cell proliferation, NF-icB activation as well as IKK-P activity. 315 [0073] Synthesis of the biotinylated DMY (DMY-biotin) [0074] Biotin (24.4 mg, 0.1 mmol) was suspended in dimethylformamide / dichloromethane (1:1, 2 mL), and dicyclohexylcarbodiimide (20.6 mg, 0.1 mmol) was added. After stirring at 60 *C for 5 minutes, dimethylaminopyridine (12.2 mg, 0.1 mmol) and DMY (48 mg, 0.15 mmol) in dimethylformamide (0.5 mL) were added. After stirring 320 overnight, the mixture was poured into water (50 mL), acidified with 3M HCI to pH 3.0, and then extracted with ethyl acetate (20 mL x3). The residue of the organic layer was subjected to silica gel chromatography (petroleum ether: acetone from 4:3 to 1:3) to 12 WO 2013/118033 PCT/IB2013/050812 afford the target product as a yellow solid (25.1 ng, 46%). Negative HR-ESI-MS: m/z 545.1203 [M-H]- (calculated for C 25
    H
    25
    N
    2 0 1 0 S: 545.1230). 325 [0075] T cell proliferation assay [00761 T lymphocyte proliferation was assessed by 5-bromo-2'-deoxy-uridine (BrdU) assay. In brief, the isolated human T lymphocytes (10' cells/well) were cultured in triplicates in a 96-well flat-bottomed plate (Costar, Corning Incorporated, Corning, NY, USA) in 100 W of RPMI 1640 medium supplemented with 10% FBS and then co 330 stimulated with P/I or OKT-3/CD28 antibodies in the presence or absence of DMY (10 100pM for 72 h. 5-bromo-2'-deoxy-uridine (BlrdU, Roche) was added to the cells 14 b before the end of stimulation at a final concentration of 10 pM. BrdU can be incorporated into the DNA of growing cells during the labeling period; the amount of BrdU incorporated into the DNA can be quantified as an indicator of cell proliferation. In this 335 experiment, BrdU was determined by ELISA according to manufacturer's instruction. [0077] NF-iB luciferase reporter assay [0078] Jurkat cells were transiently transfected with NF-B reporter plasmid with lipofectamine LTX according to the manufacturer's instructions. After transfection, cells were co-stimulated with P/I in the absence or presence of DMY or DMY-biotin for 6h. 340 Cellular proteins were lysed in Passive Lysis Buffer (Promega, Madison, WI). The transcriptional activity was determined by measuring the activity of firefly luciferase in a multi-well plate luninometer (Tecan, Durham, NC) using Luciferase Reporter Assay System (Promega). [0079] IKK- kinase assay 345 [0080] Anti-FLAG precipitated from HEK 293 expressing FLAG-IKK-$ wt, as well as the GST-IB-a substrate and ATPIMg2Cl2 were incubated in the presence or absence of DMY or DMY-biotin for lh on ice. All of the entire components were analyzed by 10% SDS-PAGE. After electrophoresis, proteins were electro-transferred to the nitrocellulose membranes. After the transfer, the membranes were blocked by 5% dried milk for 60 min 13 WO 2013/118033 PCT/IB2013/050812 350 and then washed three times (5 min in each wash) with TBS-T. The membranes were incubated with P-IxBz antibodies overnight at 4"C and then washed three times with TBS-T. Afterwards, the membranes were incubated again with HRP-conjugated secondary antibodies for 60 min. The blots were developed using the ECL. [0081] Results 355 [0082] It can be observed from Figs. 2B to 2D that DMY and DMY-biotin can inhibit T cell proliferation (Fig. 2B), NF-xB activation (Fig. 2C), as well as IKK-p kinase activity (Fig. 2D). [0083] Example 3 [0084] Study on Binding sites of IKK-p for DMY and DMY-biotin 360 [0085] This example shows that DMY directly binds to IKK-3 using DMY-biotin probe; further, DMY-biotin and DMY compound are shown to share the same binding site on IKK-p. [0086] IKK-p competition assay [0087] 20ng of human recombinant IKK-p was incubated with 100 PM of the DMY 365 biotin in the presence of 0, 1 and 5 folds of concentration of its parental compound DMY. The mixture was dropped on the nitrocellulose membranes, and then detected with streptavidin horseradish peroxidase (Sigma). The binding signal was then detected by using ECL. [0088] Results 370 [0089] As illustrated in Fig. 3, the assay shows that the parental compound DMY can compete with the biotin-DMY, indicating that the DMY-biotin is confirmed to exhibit an identical binding site(s) as its parental compound DMY. [0090] Example 4 14 WO 2013/118033 PCT/IB2013/050812 [0091] Study on Novel Binding Site(s) of IKK-p for DMY 375 [0092] This example shows that the binding site of DMY-biotin on IKK-$ is novel rather than known drug binding site(s), e.g. ATP binding site, Cys-179, Ser-177/181 and allosteric binding site. [0093] IKK-P displacement binding assay [0094] Anti-FLAG precipitated from HEK 293 expressing FLAG-IKK-$ was incubated 380 with Berberine, BMS-345541, SC-514 and BOT-64 for lh on ice, and then the mixture were incubated with 100 pM DMY-biotin. Subsequently, the proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with BSA and washing with PBS-T (Tween-20, 0.05%), the membranes were incubated with streptavidin horseradish peroxidase (Sigma) and developed with ECL. 385 [0095] Results [0096] As shown in Fig. 4, DMY binds to IKK-0 protein via novel but not well-known binding site(s). [0097] Example 5 [0098] Study on Effect of DMY on Drug Resistant Phenotype of IKK- Mutants 390 [0099] This example shows that DMY is able to circumvent the drug resistant phenotype of IKK- @ mutants with Cys-179 (C179A) and ATP-binding site (F26A) mutations. [00100] IKK-p kinase assay [00101] Anti-FLAG precipitated from HEK 293 expressing FLAG-IKK-$ C179A, F26A, as well as the GST-IxB-a substrate and ATP/Mg2Cl2 were incubated with or 395 without DMY for lh on ice. All of the components were analyzed by 10% SDS-PAGE. After electrophoresis, the proteins were electro-transferred to the nitrocellulose membranes. After the transfer, the membranes were blocked by 5% dried milk for 60 min and then washed three times (5 min in each wash) with TBS-T. The membranes were 15 WO 2013/118033 PCT/IB2013/050812 incubated with P-IBa antibodies overnight at 4*C and then washed three times with 400 TBS-T. Afterwards, the membranes were incubated again with HRP-conjugated secondary antibodies for 60 min. The blots were developed using the ECL [00102] IKK-p mutant kinase assay [00103] Anti-FLAG precipitated from HEK 293 expressing FLAG-IKK-p C179A or F26A were incubated with DMY for lh on ice, and then separated by SDS-PAGE and 405 transferred to nitrocellulose membranes. After blocking with BSA and washing with PBS-T (Tween-20, 0.05%), the membranes were incubated with streptavidin horseradish peroxidase (Sigma) and developed with ECL. [00104] Results [00105] As illustrated in Fig. 5, DMY was shown to circumvent the drug resistant 410 phenotype of IKK-P mutants with Cys-179 (C179A) and ATP-binding site (F26A) mutations. Hence, DMY was shown to bind to IKK-P via binding site(s) other than the well-known binding sites of Cys-179 residue and ATP-binding site (Phe-26). [00106] Example 6 [00107] Study on Effect of DMY on IKK- with Cysteine-46 mutation (C46A) 415 [00108] This example shows that DMY fails to suppress the kinase activity of IKK-P mutant with cysteine- 4 6 mutation (C46A), as well as fails to form protein adduct with IKK-P mutant (C46A). [00109] IKK-P kinase assay [00110] Anti-FLAG precipitated from HEK 293 expressing FLAG-IKK-j C46A, as 420 well as the GST-IB-L substrate and ATP/Mg 2 Cl 2 were incubated with or without DMY for lh on ice. All of the components were analyzed by 10% SDS-PAGE. After electrophoresis, the proteins were electro-transferred to the nitrocellulose membranes. After the transfer, the membranes were blocked by 5% dried milk for 60 mi and then 16 WO 2013/118033 PCT/IB2013/050812 washed three times (5 min in each wash) with TBS-T. The membranes were incubated 425 with P-IriBa antibodies overnight at 4*C and then washed three times with ThS-T. Afterwards, the membranes were incubated again with HRP-conjugated secondary antibodies for 60 min. The blots were developed using the ECL. [00111] IKK-$ mutant kinase assay [00112] Anti-FLAG precipitated from HEK 293 expressing FLAG-IKK-p C46A were 430 incubated with DMY for lh on ice, and then separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with BSA and washing with PBS-T (Tween-20, 0.05%), the membranes were incubated with streptavidin horseradish peroxidase (Sigma) and developed with ECL. [00113] Results 435 [00114] As seen from Fig. 6, DMY was not able to inhibit the kinase activity of IKK mutant with cysteine- 4 6 mutation (C46A), nor form protein adduct with IKK-P mutant (C46A). Hence, DMY was shown to bind to C46 residue of IKK-p. [00115] Example 7 [00116] Study on Effect of DMY on IKK-p with Cysteine Mutations 440 [00117] This example shows that DMY is able to suppress IKK-P mutants with cysteine mutations of C12A, C59A, C99A, C114A, C115A, C215A, C299A, C370A, C412A, C444A, C464A, C524A, C618A, C662/716A and C751A mutations. [00118] IKK-$ kinase assay [00119] And-FLAG precipitated from HEK 293 expressing FLAG-IKK-i wild-type (wt) 445 or mutants with cysteine mutations of C12A, C59A, C99A, C114A, C115A, C215A, C299A, C370A, C412A, C444A, C464A, C524A, C618A, C6621716A and C751A mutations as well as the GST-IB-a substrate and ATP/Mg 2
    C
    2 were incubated with or without DMY for lh on ice. All of the components were analyzed by 10% SDS-PAGE. 17 WO 2013/118033 PCT/IB2013/050812 After electrophoresis, the proteins were electro-transferred to the nitrocellulose 450 membranes. After the transfer, the membranes were blocked by 5% dried milk for 60 min and then washed three times (5 min in each wash) with TBS-T. The membranes were incubated with P-IcBa antibodies overnight at 4"C and then washed three times with TBS-T. Afterwards, the membranes were incubated again with HRP-conjugated secondary antibodies for 60 min. The blots were developed using the ECL. 455 [00120] Results [00121] As shown in Fig. 7, DMY was able to suppress IKK-$ mutants with cysteine mutations of C12A, C59A, C99A, C114A, C115A, C215A, C299A, C370A, C412A, C444A, C464A, C524A, C618A, C662/716A and C751A mutations. Hence, DMY was shown not to bind to C12 residue, C59 residue, C99 residue, C114 residue, C115 residue, 460 C179 residue, C215 residue, C299 residue, C370 residue, C412 residue, C444 residue, C464 residue, C524 residue, C618 residue, C662/C716 residue and C751 residue of IKK P3. [00122] Example 8 [00123] Study on Formation of Protein Adduct from DMY and IKK-P with Cysteine 465 Mutations [00124] This example shows that DMY is able to form protein adduct with IKK-P mutants with cysteine mutations of C12A, C59A, C99A, C1 14A, C 15A, C215A, C299A, C370A, C412A, C444A, C464A, C524A, C618A, C6621716A and C751A mutations. [00125] Protein adduct formation assay 470 [00126] Anti-FLAG precipitated from HEK 293 expressing FLAG-IKK-$ wild-type (wt) or mutants with cysteine mutations of C12A, C59A, C99A, C114A, C115A, C215A, C299A, C370A, C412A, C444A, C464A, C524A, C618A, C662/716A and C751A mutations, were incubated with DMY for lh on ice, and then separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with BSA and washing with 18 WO 2013/118033 PCT/IB2013/050812 475 PBS-T (Tween-20, 0.05%), the membranes were incubated with streptavidin horseradish peroxidase (Sigma) and developed with ECL. [00127] Results [00128] As shown in Fig. 8, DMY formed protein adduct with IKK-$ mutants with cysteine mutations, i.e. C12A, C59A, C99A, C114A, C115A, C215A, C299A, C370A, 480 C412A, C444A, C464A, C524A, C618A, C662/C716A and C751A. Hence, DMY was shown not to bind nor form protein adduct with C12 residue, C59 residue, C99 residue, C114 residue, C115 residue, C215 residue, C299 residue, C370 residue, C412 residue, C444 residue, C464 residue, C524 residue, C618 residue, C662/716 residue and C751 residue of IKK-$. 485 [00129] Example 9 [00130] Study on Effect of DMY to Suppress IKK-p - NF-B signaling [00131] This example describes that DMY is able to suppress IKK-P - NF-B signaling through Cys-46 residue of IKK-p in a cellular model. [00132] Evaluation in cellular model 490 [00133] IKK-$ -/- deficient MEFs transfected with FLAG-IKK-$ (wt) plasmid or mutant FLAG-IKK-p (C46A) plasmid were pretreated with or without 50 IiM DMY, followed by treatment of 20 ng/mL of TNF-a. The MEFs lysates were prepared for Western blotting analysis using antibodies against phosphorylation of NF-B p65 and IcBu. 495 [00134] Results [00135] As shown in Fig. 9, DMY was not able to suppress IKK-$ - NF-B signaling through Cys46 residue of IKK-p mutant (C46A) in IKK-p deficient cells model. [00136] Example 10 19 WO 2013/118033 PCT/IB2013/050812 [00137] Study on Ear Edema 500 [00138] The example shows that topically application of DMY is effective to relief mouse ear edema. [00139] The delay-type hypersensitivity test (DTHT) in mice [00140] Male ICR mice, weighting 22-30g, were obtained from the Laboratory Animal Services Center, the Chinese University of Hong Kong (Hong Kong, China). Male mice 505 were sensitized through topical application of 20pl of 0.5 % (v/v) dinitrofluoobenzene (DNFB) in acetone onto the shaved abdomen on days I and 2. Challenge was then preformed on day 6 by applying DNFB (20l1, 0.5 %, v/v) on the left inner and outer ear surfaces of mice. At the 2nd, 24th, 48th, and 72nd hour after the challenge, DMY (at doses of 0.5, 1, 2mg/ear) was topically applied (20pW) to the ears of the mice of the DMY 510 group, whereas DEX (0.025mg/ear, Sigma-Aldrich) dissolved in acetone was topically applied (20p1) to the ears of the mice of the DEX group. The mice were sacrificed by cervical dislocation, and then the same area of the ears was punched from each animal. Spleens and thymuses were isolated and weighted. The ear edema was calculated according to the differences between the weights of the right and left ears. The control 515 group was treated only with DNFB. [00141] Results [00142] The DTHT test is the reaction triggered by antigen-specific T cells that can be induced by different allergens. In this study, the most commonly used allergen, DNFB which can effectively induce the contact dermatitis on ears was used. As observed from 520 Fig. 10A, DMY could significantly and dose-dependently inhibit the ear edema of mice and the inhibition induced by of DMY was similar to the effect of DEX. [00143] Besides, as observed from from Figs. 10B and 10C, the weights of the spleen and thymus weights of the mice in the DEX group were decreased, whereas there was an increase in the weights of then spleen and thymus of the mice in the DMY group. Further, 525 the body weight of the mice in the DEX group was greatly reduced, while there was only 20 WO 2013/118033 PCT/IB2013/050812 a small decrease in the body weights of the mice in the DMY group. The difference between body weights of mice in the DMY group and the control group was not significant. [00144] In view of the above results, it can be shown that DMY suppresses 530 hypersensitivity reaction of mouse ear edema induced by DNFB. DMY is also proven to be efficacious for the treatment of dermatitis, ear inflammation, and general inflammation, without adverse effect of general immunity suppression. [00145] Example 11 [00146] Study on Arthritis 535 [00147] This example shows that DMY is effective in ameliorating collagen II induced arthritis in rats. [00148] The collagen I induced arthritis (CIA) in rats [00149] Female Wistar rats, 5-6 weeks old, were obtained from the Laboratory Animal Services Center, the Chinese University of Hong Kong (Hong Kong, China). Collagen II 540 solution (collagen, 2mg/mI in 0.05M acetic acid, Chondrex 20022, Redmond, WA, USA) was emulsified with an equal volume of incomplete Freund's adjuvant (IFA, Chondrex 7002, Redmond, WA, USA) at 4 0 C using a high-speed homogenizer. In the experiment of CIA, DMY was encapsulated with HP-CD (1:8.48) and then dissolved in the normal saline with drug concentrations of 50 and 100mg/kg body weight. Rats were 545 intradermally injected at the base of the tail with 100l collagen/ incomplete Freund's adjuvant (IFA) emulsion containing 100ig of collagen II by the use of a glass syringe equipped with a locking hub and a 27-G needle. On day 7 after the primary immunization, all the rats were given a booster injection of 100pg of collagen II in IFA. On the day after the onset of arthritis (day 13), the CIA rats were exposed to a daily intraperitoneal 550 administration of DMY (50 and 100mg/kg) until day 30 of the study. DEX (0.1 mg/kg, one per day), MTX (3.75mg/kg, twice per week), and indomethacin (1 mg/kg, one per day) were used as positive reference drugs. 21 WO 2013/118033 PCT/IB2013/050812 [00150] The rats were inspected daily from the onset of arthritis characterized by edema and/or erythema in the paws. The incidence and severity of arthritis were evaluated using 555 an arthritic scoring system, and bi-hind paw volumes and body weight were measured every 2 days starting from the day when the arthritic signs were first visible (day 13). In the arthritic scoring system, lesions (i.e. the clinical arthritic signs) of the four paws of each rat were graded from 0 to 4 according to the extent of both edema and erythema of the periarticular tissues. As such, 16 was the potential maximum of the combined arthritic 560 scores per animal. The hind paw volumes were measured using a plethysmometer chamber (7140 UGO. Basile, Comerio, Italy) and expressed as the mean volume change of both hind paws of the rats. Body weight of the rats was monitored with a 0.lg precision balance (Sartorius AG, Goettingen, Germany). On day 30, all rats were sacrificed with liver, spleen and thymus being collected and weighed. The organ index 565 for a specific organ is equal to the ratio of the weight of that organ to a body weight of 100g. [00151] Results [00152] From Figs. 11A and B, both the hind paw volume and the arthritic scores of the DMY-treated rats were significantly reduced as compared to those of the vehicle-treated 570 CIA rats. The ameliorative effect of DMY at dose of 100mg/kg (equivalent to human dose of 16mg/kg) was shown to be better than that of MTX. More importantly, it can be seen from Fig. 11 C that there was no adverse effect on the organ indexes of spleen and thymus for DMY treatment, whereas treatments with DEX, MTX, or indomethacin led to a significant reduction of the organ indexes of spleen and/or thymus. In addition, a 575 significant reduction in body weight can be observed for DEX-, MTX-, or indomethacin treated animals from Fig. 1 ID, while the DMY-treated rats were shown even to have increase in their body weights. [00153] In view of the above results, DMY was shown to suppress arthritis induced by collagen II in rats. DMY was also proven to be efficacious for the treatment of arthritis, 580 and thus inflammation, without adverse effect of general immunity suppression. The use of DMY is as described in the previous example. 22 WO 2013/118033 PCT/IB2013/050812 [00154] The exemplary embodiments of the present invention are thus fully described. Although the description referred to particular embodiments, it will be clear to one skilled in the art that the present invention may be practiced with variation of these specific 585 details. Hence this invention should not be construed as limited to the embodiments set forth herein. [00155] References 1. Keifer JA, Guttridge DC, Ashbumer BP, Baldwin AS, Jr. Inhibition of NF-kappa B activity by thalidomide through suppression of IkappaB kinase activity. I Biol Chem 590 2001;276(25):22382-7. 2. Rossi A, Kapahi P, Natoli G, et al. 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    权利要求:
    Claims (32)
    [1] 1. A method of screening a therapeutic agent as a drug candidate for cancer, inflammation, neurodegenerative disease, immunological disorder, or arthritic disorder comprising: a) exposing said agent to an assay comprising IKK-p; b) detecting whether said agent binds to cysteine-46 residue of IKK-p; c) detecting whether said agent inhibits kinase activity of IKK-p upon said binding in step (b) ; and d) identifying a drug candidate that performs said binding action of step (b) and said inhibition action of step (c).
    [2] 2. The method according to claim 1 wherein at least one binding site of IKK-p is mutated; said mutated binding site is selected from a group consisting of phenylalanine residue, serine-177/181 residue, allosteric binding site of IKK-p, and cysteine residue except cysteine-46 residue.
    [3] 3. The method according to claim 2 wherein said mutated cysteine or phenylalanine residue is selected from a group consisting of cysteine-12 residue, phenylalanine-26 residue, cysteine-59 residue, cysteine-99 residue, cysteine-114 residue, cysteine-115 residue, cysteine-179 residue, cysteine-215 residue, cysteine-299 residue, cysteine 370 residue, cysteine-412 residue, cysteine-444 residue, cysteine-464 residue, cysteine-524 residue, cysteine-618 residue, cysteine-662/716 residue, and cysteine 751 residue; said mutation is a point mutation from cysteine or phenylalanine to alanine.
    [4] 4. The method according to claim 1 wherein said cancer is selected from a group consisting of lung cancer, colon cancer, liver cancer, breast cancer, prostate cancer, cervical cancer, acute promyclocytic leukemia (APL), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma, Hodgkin's disease, chronic lymphocytic leukemia (CLL), myelodysplastic syndrome, Adult T-cell leukemia (ATL), Burkitt's lymphoma, B-cell lymphoma, primary malignant lymphocytes, B-cell chronic lymphocytic leukemia (B CLL), human THP-1 leukemia, and multiple myeloma. 29 WO 2013/118033 PCT/IB2013/050812
    [5] 5. The method according to claim 1 wherein said inflammation is selected from a group consisting of ear edema, dermatitis, ear inflammation, and arthritis.
    [6] 6. The method according to claim 1 wherein said neurodegenerative disease is selected from a group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, spinocerebellar atrophy, multiple sclerosis, and Huntington's chorea.
    [7] 7. The method according to claim 1 wherein said immunological disorder is selected from a group consisting of allergic rhinitis, allergic dermatitis, allergic contact dermatitis, allergic shock, asthma, papular urticaria, leucoderma, hypersensitivity vasculitis, hypersensitivity pneumonia, ulcerative colitis, glomerulonephritis, drug rashes, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, multiple sclerosis, hyperthyroidism, idiopathic thrombocytopenic, autoimmune hemolytic anemia, allograft rejection, and hemolytic transfusion reaction.
    [8] 8. The method according to claim I wherein said arthritic disorder is selected from a group consisting of rheumatoid arthritis, ankylosing spondylitis, gout, periarthritis, osteoarthritis, Reiter syndrome, psoriatic arthritis, post-traumatic arthritis, and enteropathic arthritis.
    [9] 9. A method for diagnosing cancer, inflammation, neurodegenerative disease, immunological disorder, or arthritic disorder in a patient comprising: a) obtaining a sample from said patient; b) contacting said sample with a compound that binds to cysteine-46 residue of IKK p of said sample; c) detecting binding of said compound to IKK-p in said sample; d) detecting inhibition action on kinase activity of IKK-p by said compound upon said binding in step (c); and e) diagnosing said patient as having a likelihood to develop cancer, inflammation, neurodegenerative disease, immunological disorder, or arthritic disorder if said compound cannot perform said binding action of step (c) and/or said inhibition action of step (d).
    [10] 10. The method according to claim 9 wherein at least one binding site of IKK-p is mutated; said mutated binding site is selected from a group consisting of 30 WO 2013/118033 PCT/IB2013/050812 phenylalanine residue, serine-177/181 residue, allosteric binding site of IKK-p, and cysteine residue except cysteine46 residue.
    [11] 11. The method according to claim 10 wherein said mutated cysteine or phenylalanine residue is selected from a group consisting of cysteine-12 residue, phenylalanine-26 residue, cysteine-59 residue, cysteine-99 residue, cysteine-114 residue, cysteine-115 residue, cysteine-179 residue, cysteine-215 residue, cysteine-299 residue, cysteine 370 residue, cysteine-412 residue, cysteine-444 residue, cysteine464 residue, cysteine-524 residue, cysteine-618 residue, cysteine-662/716 residue, and cysteine 751 residue; said mutation is a point mutation from cysteine or phenylalanine to alanine.
    [12] 12. The method according to claim 9 wherein said cancer is selected from a group consisting of lung cancer, colon cancer, liver cancer, breast cancer, prostate cancer, cervical cancer, acute promyelocytic leukemia (APL), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma, Hodgkin's disease, chronic lymphocytic leukemia (CLL), myelodysplastic syndrome, Adult T-cell leukemia (ATL), Burkitt's lymphoma, B-cell lymphoma, primary malignant lymphocytes, B-cell chronic lymphocytic leukemia (B CLL), human THP-l leukemia, and multiple myeloma.
    [13] 13. The method according to claim 9 wherein said inflammation is selected from a group consisting of ear edema, dermatitis, ear inflammation, arthritis.
    [14] 14. The method according to claim 9 wherein said neurodegenerative disease is selected from a group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, spinocerebellar atrophy, multiple sclerosis, and Huntington's chorea.
    [15] 15. The method according to claim 9 wherein said immunological disorder is selected from a group consisting of allergic rhinitis, allergic dermatitis, allergic contact dermatitis, allergic shock, asthma, papular urticaria, leucoderma, hypersensitivity vasculitis, hypersensitivity pneumonia, ulcerative colitis, glomerulonephritis, drug rashes, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, multiple sclerosis, hyperthyroidism, idiopathic thrombocytopenic, autoimmune hemolytic anemia, allograft rejection, and hemolytic transfusion reaction. 31 WO 2013/118033 PCT/IB2013/050812
    [16] 16. The method according to claim 9 wherein said arthritic disorder is selected from a group consisting of rheumatoid arthritis, ankylosing spondylitis, gout periarthritis, osteoarthritis, Reiter syndrome, psoriatic arthritis, post-traumatic arthritis, and enteropathic arthritis.
    [17] 17. A method of screening a patient to have a likelihood to develop cancer, inflammation, neurodegenerative disease, immunological disorder, or arthritic disorder comprising: a) obtaining a sample from said patient; b) contacting said sample with a compound that binds to cysteine-46 residue of IKK p of said sample; c) detecting binding of said compound to IKK-s in said sample; d) detecting inhibition action on kinase activity of IKK-p by said compound upon said binding in step (d); and e) identifying said patient as having a likelihood to develop cancer, inflammation, neurodegenerative disease, immunological disorder, or arthritic disorder if said compound cannot perform said binding action of step (c) and/or said inhibition action of step (d).
    [18] 18. The method according to claim 17 wherein at least one binding site of IKK-p is mutated; said mutated binding site is selected from a group consisting of phenylalanine residue, serine-177/181 residue, allosteric binding site of IKK-s, and cysteine residue except cysteine-46 residue.
    [19] 19. The method according to claim 18 wherein said mutated cysteine or phenylalanineresidue is selected from a group consisting of cysteine-12 residue, phenylalanine-26 residue, cysteine-59 residue, cysteine-99 residue, cysteine-114 residue, cysteine-115 residue, cysteine-179 residue, cysteine-215 residue, cysteine 299 residue, cysteine-370 residue, cysteine-412 residue, cysteine-444 residue, cysteine464 residue, cysteine-524 residue, cysteine-618, cysteine-662/716 residue, and cysteine-751 residue; said mutation is a point mutation from cysteine or phenylalanine to alanine.
    [20] 20. The method according to claim 17 wherein said cancer is selected from a group consisting of lung cancer, colon cancer, liver cancer, breast cancer, prostate cancer, cervical cancer, acute promyelocytic leukemia (APL), acute myeloid leukemia 32 WO 2013/118033 PCT/IB2013/050812 (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma, Hodgkin's disease, chronic lymphocytic leukemia (CLL), myelodysplastic syndrome, Adult T-cell leukemia (ATL), Burkitt's lymphoma, B-cell lymphoma, primary malignant lymphocytes, B-cell chronic lymphocytic leukemia (B CLL), human THP-1 leukemia, and multiple myeloma.
    [21] 21. The method according to claim 17 wherein said inflammation is selected from a group consisting of ear edema, dermatitis, ear inflammation, arthritis.
    [22] 22. The method according to claim 17 wherein said neurdegenerative disease is selected from a group consisting of Alzheimers disease, Parkinson's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, spinocerebellar atrophy, multiple sclerosis, and Huntington's chorea.
    [23] 23. The method according to claim 17 wherein said immunological disorder is selected from a group consisting of allergic rhinitis, allergic dermatitis, allergic contact dermatitis, allergic shock, asthma, papular urticaria, leucoderma, hypersensitivity vasculitis, hypersensitivity pneumonia, ulcerative colitis, glomerulonephritis, drug rashes, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, multiple sclerosis, hyperthyroidism, idiopathic thrombocytopenic, autoimmune hemolytic anemia, allograft rejection, and hemolytic transfusion reaction.
    [24] 24. The method according to claim 17 wherein said arthritic disorder is selected from a group consisting of rheumatoid arthritis, ankylosing spondylitis, gout, periarthritis, osteoarthritis, Reiter syndrome, psoriatic arthritis, post-traumatic arthritis, and enteropathic arthritis.
    [25] 25. A method for treating cancer, inflammation, neurodegenerative disease, immunological disorder, or arthritic disorder comprising administering an effective amount of a therapeutic agent to a patient in need thereof, wherein said patient harbors gene mutations on at least one binding site of IKK-p; said mutated binding site is selected from a group consisting of phenylalanine residue, serine-177/181 residue, allosteric binding site of IKK-p, and cysteine residue except cysteine-46 residue.
    [26] 26. The method according to claim 25 wherein said mutated cysteine or phenylalanineresidue is selected from a group consisting of cysteine-12 residue, 33 WO 2013/118033 PCT/IB2013/050812 phenylalanine-26 residue, cysteine-59 residue, cysteine-99 residue, cysteine-114 residue, cysteine-115 residue, cysteine-179 residue, cysteine-215 residue, cysteine 299 residue, cysteine-370 residue, cysteine-412 residue, cysteine-444 residue, cysteine-464 residue, cysteine-524 residue, cysteine-618 residue, cysteine-662 /716 residue, and cysteine-751 residue; said mutation is a point mutation from cysteine or phenylalanine to alanine.
    [27] 27. The method according to claim 25 wherein said therapeutic agent binds to cysteine-46 residue of IKK-p and inhibits the kinase activity of IKK-p upon said binding.
    [28] 28. The method according to claim 25 wherein said cancer is selected from a group consisting of lung cancer, colon cancer, liver cancer, breast cancer, prostate cancer, cervical cancer, acute promyelocytic leukemia (APL), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma, Hodgkin's disease, chronic lymphocytic leukemia (CLL), myelodysplastic syndrome, Adult T-cell leukemia (ATL), Burkitt's lymphoma, B-cell lymphoma, primary malignant lymphocytes, B-cell chronic lymphocytic leukemia (B CLL), human TIP-I leukemia, and multiple myeloma.
    [29] 29. The method according to claim 25 wherein said inflammation is selected from a group consisting of ear edema, dermatitis, ear inflammation, arthritis.
    [30] 30. The method according to claim 25 wherein said neurodegenerative disease is selected from a group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, spinocerebellar atrophy, multiple sclerosis, and Huntington's chorea.
    [31] 31. The method according to claim 25 wherein said immunological disorder is selected from a group consisting of allergic rhinitis, allergic dermatitis, allergic contact dermatitis, allergic shock, asthma, papular urticaria, leucoderma, hypersensitivity vasculitis, hypersensitivity pneumonia, ulcerative colitis, glomerulonephritis, drug rashes, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, multiple sclerosis, hyperthyroidism, idiopathic thrombocytopenic, autoimmune hemolytic anemia, allograft rejection, and hemolytic transfusion reaction.
    [32] 32. The method according to claim 25 wherein said arthritic disorder is selected from a group consisting of rheumatoid arthritis, ankylosing spondylitis, gout, periarthritis, 34 WO 2013/118033 PCT/IB2013/050812 osteoarthritis, Reiter syndrome, psoriatic arthritis, post-traumatic arthritis, and enteropathic arthritis. <00094202-RM> 35
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    同族专利:
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    WO2013118033A1|2013-08-15|
    US20150031050A1|2015-01-29|
    US20130210875A1|2013-08-15|
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    法律状态:
    2017-02-09| MK1| Application lapsed section 142(2)(a) - no request for examination in relevant period|
    优先权:
    申请号 | 申请日 | 专利标题
    US13/370,313|US20130210875A1|2012-02-10|2012-02-10|Novel binding site of IKK-beta|
    US13/370,313||2012-02-10||
    PCT/IB2013/050812|WO2013118033A1|2012-02-10|2013-01-31|NOVEL BINDING SITE OF ΙΚΚ-β|AU2013101130A| AU2013101130A4|2012-02-10|2013-08-22|Novel binding site of ikk beta|
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