EP0765475A1 - Triage pour la detection de modulateurs des cytokines - Google Patents

Triage pour la detection de modulateurs des cytokines

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Publication number
EP0765475A1
EP0765475A1 EP95921363A EP95921363A EP0765475A1 EP 0765475 A1 EP0765475 A1 EP 0765475A1 EP 95921363 A EP95921363 A EP 95921363A EP 95921363 A EP95921363 A EP 95921363A EP 0765475 A1 EP0765475 A1 EP 0765475A1
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European Patent Office
Prior art keywords
promoter
protein
rel
binding
receptor
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EP95921363A
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German (de)
English (en)
Inventor
Thomas Hermann
John W. Pike
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Ligand Pharmaceuticals Inc
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Ligand Pharmaceuticals Inc
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    • 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
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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

Definitions

  • This invention relates to a method for screening for agents useful for treatment of diseases and pathological conditions affected by cytokines and novel agents identified using such screening method.
  • Cytokines are a group of molecules capable of signalling cellular development. Aberrant expression of cytokines is known to be associated with pathological conditions including autoimmune diseases, septic shock, rheumatoid arthritis, psoriasis, inflammation, postmenopausal osteoporosis, and some cancers. Common treatment for these pathological conditions are retinoids, immunosuppressants, glucocorticoids and other steroid drugs. Estrogens are specifically employed in the prevention of postmenopausal osteoporosis. Steroids and related hormone drugs exert their therapeutic effects by binding to a superfamily of intracellular receptors (IRs) , which are regulators of gene transcription. IRs can function as activators as well as repressors of specific cytokine genes. The activity of IRs is controlled by hormones or other ligands that bind to the IRs.
  • IRs intracellular receptors
  • IRs The classical mechanism of transcriptional regulation by IRs involves binding of the IRs to specific response elements in the promoters of the regulated genes, for example, the binding of the estrogen receptor to its response site in the vitellogenin gene (Klein-Hitpass et al. , Cell 46:1053- 1061, 1986) . More recently a different mechanism of IRs function has been described in glucocorticoid receptor mediated AP-1 transcription regulation that does not require direct DNA-binding of the IRs (Yang-Yen et al. , Cell 62:1205-1215, 1990) .
  • steroid drugs have been shown to repress the level of certain cytokines, a lack of tissue specificity and side effects of the steroids may limit their use as therapeutic agents. These side effects may be reduced or completely avoided with more specifically acting compounds.
  • Pfahl and Karin (PCT publication, WO 92/07072, 1992) describes a method of screening a sample for ligands which bind to a nuclear receptor to form a complex which binds or interferes with a non rel-like protein AP-1 or an AP-1 component.
  • the present invention relates to a method for identifying new therapeutic agents and for using these agents to treat diseases and conditions affected by cytokines, such as, but not limited to, osteoporosis, rheumatoid arthritis, inflammation, psoriasis, septic shock, Kaposi's sarcoma and multiple myeloma.
  • cytokines such as, but not limited to, osteoporosis, rheumatoid arthritis, inflammation, psoriasis, septic shock, Kaposi's sarcoma and multiple myeloma.
  • cytokine is generally meant a secreted protein which acts as a chemical mediator of cellular regulation. More specifically, it is meant a diverse groups of soluble polypeptides such as growth factors and hormones that control the growth, differentiation and function of cells, including, but not limited to, GM-CSF, G-CSF, IL-2, IL-6, IL-8, and IL-11.
  • the present invention relates to the determination that inhibition of interleukin 6 (IL-6) expression by estrogen-estrogen receptor complex is mediated through the control of the transcriptional activity of NFKB or closely related proteins on the IL-6 promoter.
  • This mechanism does not involve direct binding of ER to IL-6 promoter but controls the DNA-binding properties of the activated NF/cB and possible other members of the rel- family of proteins to their specific response elements (i.e., rel site) on the IL-6 promoter.
  • NF/cB is involved in the regulation of genes encoding various cytokines and their receptors, viral proteins, and proteins involved in the acute-phase response
  • the regulation of NF/cB activity by estrogen and possible other hormones is of general importance (see generally Baeuerle, Biochemica et Biophysica Acta, 1072:63-80, 1993, incorporated by reference herein) .
  • retinoic acid treatment which strongly inhibits I -6 expression in +/+LDA11 cells and other tissues (Gross, V., P. M. Villiger, B. Zhang, and M. Lotz, 1993, "Retinoic acid inhibits interleukin-1- induced cytokine synthesis in human monocytes," J . Leukoc.
  • Biol. 54:125-132 has the same effect as estrogen on the NF/cB related complexes with the IL-6 promoter. This suggests a general pathway of transcriptional regulation involving cross-talk between members of the intracellular receptor family and the NF/cB transcription factors.
  • the above determination allows for the screening of drugs that specifically influence genes controlled by the rel-transcription factors, i.e. genes involved in inflammation, sepsis, skin and kidney disorders, osteoporosis, certain cancers, and hematopoietic dysfunctions without the side effects of known steroid drugs.
  • the diseases listed are usually correlated with aberrant expression of cytokines such as IL-1, TNFo., IL- 6, IL-8 that are under the control of NF/cB or other rel proteins.
  • the present invention features a method for identifying agents which, by activating an intracellular receptor, cause a significant reduction in the binding of a rel-like protein or other transcriptional protein to the rel site on the promoter of a cytokine gene or a portion of the promoter, thereby reducing the transcription of the cytokine.
  • intracellular receptor an intracellular transcription factor whose activity is regulated by binding of small molecules, including, but not limited to, estrogen receptor, retinoid acid receptors, retinoid X receptors, glucocorticoid receptor, progesterone receptors, androgen receptor, thyroid hormone receptors, and vitamin D receptor.
  • Rel-like protein is meant a protein or a protein complex of the rel family that share a homology in the rel domain and is involved in gene regulation (see Liou and Baltimore, Current Opinion in Cell Biology, 5:477-487, 1993, incorporated by reference herein) , including, but not limited to, NF/cB, Lyt-10, c- rel, and relB.
  • transcriptional protein is meant a cytoplasmic or nuclear protein that, when activated, bind a promoter either directly, or indirectly through a complex of proteins to modulate the transcription activity of the promoter.
  • rel site is meant a DNA sequence that serves as a binding site for rel-like proteins or complexes comprising one or more rel-like proteins, including, but not limited to, /_B motifs identified in Baeuerle, Biochemica et Biophysica Acta, 1072:63-80, 1993, incorporated by reference herein, such as the NF/cB binding site on IL-6 promoter.
  • promoter is meant a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • a promoter of a DNA construct, including an oligonucleotide sequence according to the present invention may be linked to a heterologous gene when the presence of the promoter influences transcription from the hete'rologous gene, including genes for reporter sequences such as luciferase, chloramphenicol acetyl transferase, ⁇ - galactosidase and secreted placental alkaline phosphatase.
  • the assay is conducted in a whole cell system that has an intracellular receptor which is the target of the screened agent, a promoter or a portion of a promoter with a rel site and a rel-like protein or other transcription protein that binds to the rel site; wherein the intracellular receptor modulates the binding of the rel-like protein or the transcription protein to the rel site.
  • the intracellular receptor, the promoter or a portion of the promoter, or the protein that binds to the rel site may either be endogenous to the cell or transfected into the cell.
  • the assay is conducted in an extract of cell having an intracellular receptor, a promoter or a portion of a promoter, with a rel site and a rel-like protein or other protein that binds to the rel site; wherein the intracellular receptor modulates the binding of the rel-like protein or the transcription protein to the rel site.
  • the binding of the rel-like protein or other transcription protein to the rel site may be measured by techniques known to those skilled in the art, including, but not limited to, mobility shift assay, co- transfection assay, and expression of a reporter gene linked to the promoter.
  • the promoter is activated by an effector, including, but not limited to, tumor necrosis factor, interleukin-1, viruses, endotoxins, phorbol esters, epidermal growth factor, leukemia inhibitor factor and cAMP agonists.
  • an effector including, but not limited to, tumor necrosis factor, interleukin-1, viruses, endotoxins, phorbol esters, epidermal growth factor, leukemia inhibitor factor and cAMP agonists.
  • effector is meant an agent that stimulates the expression of a cytokine to a measurable level .
  • An effector may be endogenously produced in a cell or exogenously added to a cell
  • the claimed assay is conducted in a system including an estrogen receptor, an interleukin 6 promoter or a portion of an IL-6 promoter and NF/cB; wherein ER modulates the binding of NF/cB or related proteins to the NF/cB site on the IL-6 promoter.
  • agents discovered by the above assay may either interact directly with an intracellular receptor, or modulate the interaction of a ligand with the intracellular receptor.
  • a ligand for the intracellular receptor is included in the assay.
  • steroids and steroid analogs may exemplify agents identified by the present invention
  • Applicant is particularly interested in the identification of agents of low molecular weight (less than 10,000 daltons, preferably less than 5,000, and most preferably less than 1,000) which can be readily formulated as useful therapeutic agents.
  • agents can then be screened to ensure that they are specific to tissues with cytokine inflicted pathological conditions with little or no effect on healthy tissues such that the agents can be used in a therapeutic or prophylactic manner. If such agents have some effect on healthy tissues they may still be useful in therapeutic treatment, particularly in those diseases which are life threatening, such as Kaposi's sarcoma or multiple myeloma. Once isolated, a candidate agent can be put in pharmaceutically acceptable formulations, and used for specific treatment of diseases and pathological conditions with little or no effect on healthy tissues.
  • Figure 1 shows IL-1 and TNF ⁇ induced complex formation on the proximal IL-6 promoter.
  • Figure 2 shows that several distinct NF/cB-related complexes induced by IL-1 and TNFo. are modulated by estrogen.
  • Figure 3 shows the effects of estrogen agonist and antagonist, and inhibitors of protein synthesis and protein kinase C on the formation of NF/cB-related complexes.
  • Figure 4 shows the binding characteristics of proteins in NF/cB-related complexes with NF/cB oligonucleotides.
  • Figure 5 shows NF/cB related proteins in complexes A,
  • cytokines including IL-6, 11-8 and IL- 11, have related biological effects, i.e., effects on cellular defense in response to infection by stimulating the immune and the acute-phase response and on bone metabolism by increasing bone resorption. Aberrant expression of any of these cytokines results in similar pathological conditions, e.g., all cytokines listed are involved in septic shock. In another example, excessive production IL-8, like IL-6, may be involved in the pathogenesis of several types of inflammatory reactions, particularly neutrophil-dependent tissue damages. These cytokines have similar promoter structures, e.g., their promoters contain binding sites for NF/cB or other rel proteins. It is therefore likely that not only IL-6 but also the other cytokines mentioned above can be targeted by drugs that modulate the binding of NF/cB or other rel proteins to their promoter sites through the intracellular receptors.
  • Interleukin-6 is a pleiotropic cytokine that is secreted by many different cells, including monocytes, macrophages, certain B-lymphocytes and T- lymphocytes, glial cells, fibroblasts, osteoblasts, and stromal cells (reviewed in references Hirano, T. , (1992) "The biology of interleukin-6, " Chem. Immunol. 51:153- 180.; Kishimoto, T. (1989) "The biology of interleukin- 6," Blood 74:1-10.; Kishimoto, T., M. Hibi, M. Murakami, M. Narazaki, M. Saito, and T.
  • IL-6 expression is usually associated with disease (Yu, X.P., T. Bellido, N. Rice, and S.C. Manolagas (1993) .
  • IL-6 expression is tightly controlled by other factors. Depending on the particular cell type, it can be activated by various stimuli, including tumor necrosis factor (TNFc.) and interleukin-1 (IL-1) , viruses, endotoxin (lipopolysaccharides) , phorbol esters, epidermal growth factor (EGF) , leukemia inhibitor factor (LIF) , and cAMP agonists.
  • TNFc. tumor necrosis factor
  • IL-1 interleukin-1
  • viruses include endotoxin (lipopolysaccharides) , phorbol esters, epidermal growth factor (EGF) , leukemia inhibitor factor (LIF) , and cAMP agonists.
  • ENF epidermal growth factor
  • LIF leukemia inhibitor factor
  • IL-6 promoter By sequence comparison several potential transcriptional control elements have been identified in the IL-6 promoter, including a cAMP response element, an AP-1 binding site, and binding elements for the transcription factors NF-IL6 (C/EBPB, LAP, AGP/EBP) and NF/cB (Isshiki, H. , S. Akira, 0. Tanabe, T. Nakajima, T. Shimamoto, T. Hirano, and T. Kishimoto (1990) "Constitutive and interleukin-1 (IL-1) - inducible factors interact with the IL-1-responsive element in the IL-6 gene," Mol. Cell Biol. 10:2757- 2764) .
  • NF-IL6 nuclear factor for IL-6 expression
  • NF-IL6 belongs to the C/EBP family of leucine zipper proteins.
  • IL-6 lipopolysaccharide
  • NF-IL6 nuclear factor for IL-6 expression
  • NF-IL6 nuclear factor for IL-6 expression
  • AGP/EBP transcription factor
  • Mol. Cell Biol. 10:6642-6653 Descombes, P., M. Chojkier, S. Lichtsteiner, E. Falvey, and U.
  • LAP a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein
  • Genes Dev. 4:1541-1551 Descombes, P., M. Chojkier, S. Lichtsteiner, E. Falvey, and U. Schibler (1990) "LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein," Genes Dev. 4:1541-1551; Poli, V., F.P.
  • IL-6DBP a nuclear protein involved in interleukin-6 signal transduction
  • C/EBP C/EBP
  • IL-6DBP a nuclear protein involved in interleukin-6 signal transduction
  • NF/cB is a transcription factor that was originally identified as a heterodimeric complex consisting of a 50 kD protein (p50) and a 65 kd protein (p65) that binds an element in the immunoglobulin kappa light chain enhancer.
  • Both proteins reveal a high homology to the Drosophila morphogen dorsal and to the c-rel proto-oncogeny product.
  • the p65 subunit is also functionally related to c-rel (reviewed in references Baeuerle, P. A. (1991) "The inducible transcription activator NF-kappa B: regulation by distinct protein subunits” Biochim. Biophvs. Acta 1072:63-80; Blank, V., P. Kourilsky, and A. Israel (1992) "NF-kappa B and related proteins: Rel/dorsal homologies meet ankyrin- like repeats," Trends. Biochem. Sci. 17:135-140; and Liou, H.C. and D.
  • NF/cB a new Rel family transcription activator that can interact with p50-NF-kappa B, " Mol. Cell Biol. 12:674-684; Schmid, R.M. , N.D. Perkins, C.S. Duckett, P.C. Andrews, and G.J. Nabel (1991) "Cloning of an NF-kappa B subunit which stimulates HIV transcription in synergy with p65," Nature 352:733-736) .
  • NF/cB is located in the cytosol complexes with an inhibitory protein of the I/cB family.
  • NF/cB Upon induction, NF/cB dissociates from I/cB and translocates into the nucleus where it binds and activates specific promoters (Baeuerle, P.A. and D. Baltimore (1988) "I kappa B: a specific inhibitor of the NF-kappa B transcription factor," Science 242:540-546; Ghosh, S. and D. Baltimore (1990) "Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B, " Nature 344:678-682) .
  • Binding of NF/cB-like factors to the consensus site of • the IL-6 promoter is induced by IL-1, TNFo., LIF, LPS and phorbol esters, varying with the particular cell type (Gruss, H.J., M.A. Brach, and F. Herrmann (1992) "Involvement of nuclear factor-kappa B in induction of the interleukin-6 gene by leukemia inhibitory factor," Blood 80:2563-2570; Libermann, T.A. and D. Baltimore (1990) "Activation of interleukin-6 gene expression through the NF-kappa B transcription factor," Mol. Cell Biol. 10:2327-2334; Shimizu, H., K. Mitomo, T.
  • mice that carry a null mutation for IL-6 do not affect bone volume or osteoclast number as seen with normal mice (Balena, R. , F. Costantini, M. Yamamoto, A. Markatos, R. Cortese, G.A. Rodan, and V. Poli (1993) "Mice with IL-6 gene knock-out do not lose cancellous bone after ovariectomy, " J. Bone Miner. Res. 8:S130 [Abstract]) .
  • Estrogen has been found to inhibit IL-6 expression in bone-derived stromal cell lines and osteoblastic cells from rats and mice as well as in nontransformed human bone cells (Girasole, G., R.L. Jilka, G. Passeri, S. Boswell, G. Boder, D.C. Williams, and S.C. Manolagas (1992) "17 beta-estradiol inhibits interleukin-6 production by bone marrow-derived stromal cells and osteoblasts in vitro: a potential mechanism for the antiosteoporotic effect of estrogens," J. Clin. Invest. 89:883-891) .
  • oligonucleotide covering the potential NF/cB site of the IL-6 promoter competed against the induced binding to this fragment, while an oligonucleotide covering the NF-IL6 site was ineffective.
  • the NF/cB oligonucleotide was used as probe, three IL-1/TNFo.- induced complexes were observed.
  • Pretreatment with estradiol decreased the intensity of the slowest complex and strongly increased the intensity of the fastest migrating complex.
  • the three bands were differentially supershifted (i.e., further decrease in the mobility of the complex due to binding of the antibody) by anti-p50 and anti-p65 antibodies, while none of several other antibodies tested, including anti-ER antibody, had any effect.
  • Methylation interference assays showed identical DNA contact sites for all three complexes.
  • a candidate agent will be screened by either A) direct evaluation of protein binding to rel-sites, or B) indirect evaluation of binding to rel-sites.
  • Cells selected for expression of the necessary components will be treated with the agent or vehicle control and an inducer (e.g., phorbol ester, cytokines, lipopolysaccharides) .
  • an inducer e.g., phorbol ester, cytokines, lipopolysaccharides
  • Cellular extracts prepared from those cells e.g., whole cell, cytosolic, or nuclear extracts
  • Binding will be analyzed qualitatively (i.e., comparing pattern) and quantitatively comparing extracts from cells treated with vehicle or the agent.
  • Cells selected for expression of the necessary components and their production of cytokine will be treated with the agent or vehicle control and an inducer (phorbol ester, cytokines, lipopolysaccharides) .
  • an inducer phorbol ester, cytokines, lipopolysaccharides
  • Activity of the agent will be quantitatively assessed by measuring of cytokine using standard assays known to those skilled in the art.
  • a reporter construct By means of transfection a reporter construct will be introduced into the cells that expresses an easily measurable protein under the control of a cytokine promoter or fragments thereof or isolated rel-sites.
  • the other necessary components are either expressed endogenously by the cells or provided by cotransfection of expression vectors for the particular component.
  • Cells will be treated with the agent or vehicle control and an effector (phorbol ester, cytokines, lipopolysaccharides) .
  • the activity of the agent will be analyzed quantitatively by measuring the expression of the reporter protein.
  • Agents will also be tested for their binding to IRs by traditional binding assays as well as for their activity to effect the classical mechanism of gene regulation by IRs.
  • An agent that binds to IRs and regulates binding of rel proteins to cytokine promoters but does not activate the classical mechanism of IR action is a potential drug candidate for the specific treatment of diseases associated with aberrant expression of cytokines.
  • the pIL6 [-225] Luc reporter construct was derived from the parental pIL6 [- 1200]Luc by excision of a Nhel-BamHI fragment and religation of the vector fragment after blunt ending with Klenow DNA-polymerase.
  • the parental pIL6 [-1200] Luc was constructed by cloning the 1.2 kb IL-6 promoter insert excised with Ba HI and Kpnl from pCAT-M54-IL6 (-) into the corresponding sites of the luciferase vector Lucpl.
  • C3H10T1/2 cells were seeded in phenol-red-free DMEM supplemented with 10% FBS at 80,000 cells per well (12- well plates) .
  • the cells were transfected by calcium phosphate precipitation (Peterson, J.L. and O.W. McBride (1980) "Cotransfer of linked eukaryotic genes and efficient transfer of hypoxanthine phosphoribosyltransferase by DNA-mediated gene transfer," Proc. Natl. Acad. Sci. U.S.A.
  • IL-lb (1 nM each) for 24 h. After a brief wash with PBS the cells were lysed in 200 ml lysis buffer (25 mM Tris [pH 7.8] , 2 mM DTT, 2 mM 1, 2-diaminocyclohexane- N,N,N' ,N' -tetraacetic acid, 10% glycerol, 1% Triton X- 100) .
  • lysis buffer 25 mM Tris [pH 7.8] , 2 mM DTT, 2 mM 1, 2-diaminocyclohexane- N,N,N' ,N' -tetraacetic acid, 10% glycerol, 1% Triton X- 100
  • Peptides used to raise the following antibodies in rabbits correspond to amino acid residues 91-105 of murine c-jun (Ryder and Nathans (1988) "Induction of protooncogene c-jun by serum growth factors," Proc.
  • Anti-TBP was a protein-A purified serum preparation from a rabbit immunized with the full length human recombinant protein and reported to react with TBP from mouse, rat, and human origin (Santa Cruz Biotechnology, Inc.) .
  • Anti-ER is a mouse monoclonal antibody (IgG2a) raised against a peptide corresponding to amino acid residues 8-22 of the murine ER.
  • IL-6 concentration in tissue culture supernatants was determined by use of an IL-6 ELISA kit (Endogen, Inc., Boston, MA) using murine IL-6 as standard.
  • ER in +/+LDA11 cells was measured in whole cell extracts.
  • Electrophoretic mobility shift assay (EMSA) and methylation interference assay
  • DNA binding studies were carried out with nuclear extracts from +/+LDA11 cells, extracts from yeast expressing recombinant human ER gly , and purified p50 and p49 proteins.
  • +/+LDA11 cells were maintained under conditions as described (Girasole, G. , R.L. Jilka, G.
  • Lysates were transferred into microfuge tubes, nuclei pelleted (8000 rpm, 1 min) and resuspended in buffer C (20 mM HEPES [pH 7.9] , 1.5 mM MgCl 2 , 420 mM NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF) . After 40 min rocking at 4 °C, samples were centrifuged (15,000 rpm, 10 min) and supernatants taken as nuclear extracts.
  • Bradford protein assays (Bradford, M.M. (1976) "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding," Anal.
  • Extracts of yeast recombinantly expressing ER gly were prepared from the BJ2168 strain transformed with YEpElO as described (Tzukerman, M. , A. Esty, D. Santiso-Mere, P. Danielian, M.G. Parker, R.B. Stein, J.W. Pike, and D.P. McDonnell (1994) "Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions, " Mol. Endocrinol. 8:21-30) .
  • Probes were either double stranded oligonucleotides corresponding to the regions -82 to -47
  • Example 1 Screening for ER mediated inhibition of IL-6 promoter activity
  • C3H10T1/2 cells were transfected with a luciferase expression vector under the control of the proximal human IL-6 promoter (pIL6 [-225] Luc) alone or together with the expression vector for the wild-type human ER gly (pRShER) .
  • the cultures were induced with 1 nM each of TNFc. and IL-1 or left uninduced and 24 h later cells were harvested and extracts analyzed for luciferase activity.
  • Treatment of transfected cells with IL-1 and TNFc. induced a 5-fold increase in luciferase activity over basal levels.
  • C3H10T1/2 cells were transfected with a luciferase expression vector under the control of the minimal thymidine kinase promoter and the vitellogenin estrogen response element (pERE-tk-Luc) alone or together with pRShER. 24 h after treatment with 10 nM estradiol or vehicle cells were harvested and extracts analyzed for luciferase activity. Induction of luciferase activity by estradiol was only observed in the presence of cotransfected ER.
  • C3H10T1/2 cells were incubated with or without 10 nM estradiol. After 24 h the cultures were induced with TNFc. and IL-1 (1 nM each) or left uninduced for additional 24 h. 11-6 in the supernatants was assayed by an ELISA specific for murine IL-6.
  • C3H10T1/2 cells responded to IL-1 and TNFc. treatment with strongly increased production of endogenous IL-6, but unlike other osteogenic or stromal cells containing endogenous ER (Girasole, G., R.L. Jilka, G. Passeri, S. Boswell, G. Boder, D.C Williams, and S.C.
  • Example 2 Screening agents that modulates binding of NFcB related proteins to the proximal IL-6 promoter
  • An exemplary assay system is a cell line that expressed all the necessary components endogenously, including the ER.
  • the bone marrow derived murine stromal cell line +/+LDA11 has been shown to respond to IL-1 and TNF ⁇ treatment with strongly increased secretion of IL-6.
  • Treatment with estradiol inhibits this induction of IL-6 as shown for the protein and its mRNA (Girasole, G. , R.L. Jilka, G. Passeri, S. Boswell, G. Boder, D.C. Williams, and S.C.
  • ER-specific DNA binding activity could not be detected in nuclear extracts from +/+LDA11 treated with estradiol and/or IL-1 and TNFc.
  • Nuclear extracts of +/+LDA11 cells pretreated with estradiol (10 nM) and TNFc. and IL-1 (InM each for 40 min) as indicated or yeast extract containing recombinantly expressed human wild-type ER gly were incubated with the vitellogenin ERE as probe in the absence or presence of anti-ER antibody. Complexes formed were analyzed by EMSA.
  • IL-6 induction nuclear extracts from +/+LDA11 cells were analyzed for DNA-binding activity to the IL-6 promoter region that mediated cytokine induction and estrogen suppression in the cotransfection experiments. Since this DNA fragment showed a high background binding with nuclear extracts the most proximal region containing the TATA box was removed leaving a fragment from -225 to -52 upstream of the transcriptional start site.
  • This region of the promoter contains consensus binding sites for several transcription factors including a core sequence of the cAMP response element (CRE) , a binding site for the leucine zipper protein NF-IL6, and a NF/cB site (Isshiki, H., S. Akira, 0.
  • CRE cAMP response element
  • NF-IL6 nuclear factor for IL-6 expression
  • NF-IL6 nuclear factor for IL-6 expression
  • EMBO J. 9:1897-1906 EMBO J. 9:1897-1906
  • Libermann, T.A. and D. Baltimore (1990) Activation of interleukin-6 gene expression through the NF-kappa B transcription factor, " Mol. Cell Biol. 10:2327-2334) .
  • This fragment was incubated with nuclear extracts from +/+LDA11 cells that had been treated with IL-1 and TNF ⁇ for various times. +/+LDA11 cells were pretreated with 10 nM estradiol as indicated. After 24 h the cells were induced with TNF ⁇ and IL-1 (1 nM each) for various periods of time. Induction was stopped by cell lysis and nuclear extracts were analyzed by EMSA using the -225 to -52 IL-6 promoter fragment as probe ( Figure la) .
  • estradiol pretreatment had no effect on the binding capacity of extracts from uninduced cells.
  • estradiol pretreatment resulted in a marked increase of the induced complex with induction intervals from 10 min to 40 min but only a slight effect on the complex after 2 h of induction.
  • pretreatment with estradiol also caused a qualitative change, increasing the mobility of the complex.
  • Fig. lb shows that none of the antibodies tested affected DNA binding of extracts from uninduced cells. Neither anti-c-jun, nor anti-c-rel, nor anti-NF-IL6 antibodies had any effect on the cytokine induced complexes.
  • Fig. lc shows that independent of cytokine induction or estradiol treatment, addition of the anti-ER antibody did not significantly affect any of the complexes, induced or constitutive.
  • yeast extracts containing high concentrations of recombinant ER were incubated with the IL-6 promoter fragment no specific binding of the ER was detected (lanes 7 and 8) .
  • the weak bands observed are unrelated to the ER, since they were not affected by the anti-ER antibody.
  • IL-1 and TNF ⁇ specifically activated NF/cB or related proteins. No binding of c-jun (AP-1) , NF-IL6, c-rel, or ER was detected.
  • NF-IL6 binding is surprising, since induction and binding of this transcription factor in response to IL-1 has been reported for other cells (Akira, S., H. Isshiki, T. Sugita, 0. Tanabe, S. Kinoshita, Y. Nishio, T. Nakajima, T. Hirano, and T.
  • NF-IL6 nuclear factor for IL-6 expression
  • NF-IL6 nuclear factor for IL-6 expression
  • c-rel activates but v-rel suppresses transcription from kappa B sites
  • Proc. Natl. Acad. Sci. USA 88:3715-3719 Our DNA binding experiments show that in the bone marrow derived +/+LDA11 cell IL-1 and TNF ⁇ induce the binding of NF/cB or closely related proteins to the IL-6 promoter.
  • AP-1 a transcription factor
  • This transcription factor is one of the paradigms for direct inhibition by intracellular receptor including GR, RAR and TR.
  • the mechanism of AP-1 inhibition can involve protein-protein interaction and/or competition for DNA binding depending on the particular gene (Diamond, M.I., J.N. Miner, S.K. Yoshinaga, and K.R. Yamamoto (1990) "Transcription factor interactions: selectors of positive or negative regulation from a single DNA element," Science 249:1266-1272; Sch ⁇ le, R. , K. Umesono, D.J. Mangelsdorf, J. Bolado, J.W.
  • Example 3 Screening agents that differentially affect distinct complexes with the IL-6 promoter
  • Distinctive complexes with the IL-6 promoter Treatment of +/+LDA11 cells with IL-1 and TNF ⁇ induced binding of NF/cB or related proteins to the IL-6 promoter. Since pretreatment with estradiol not only increased the intensity but also the mobility of the complexes, we investigated the binding of +/+LDA11 nuclear extracts to the oligonucleotide covering the putative NF/cB site (-82 to -47) .
  • +/+LDA11 cells were pretreated with 10 nM estradiol as indicated. After 24 h the cells were induced with TNF ⁇ and IL-1 (1 nM each) for various periods of time. Induction was stopped by cell lysis and nuclear extracts were analyzed by EMSA using the -82 to -47 IL-6 promoter fragment as probe.
  • Fig. 2a shows that extracts from cells treated with IL-1 and TNF ⁇ exhibited 3 induced complexes (A,B,C) when compared with extracts from untreated cells. Over the course of induction (10-120 min) in particular the fastest migrating complex (C) decreased in intensity.
  • estradiol pretreatment reduced the intensity of the slowest migrating complex (A) while strongly increasing the intensity of the fastest band (C) .
  • Nuclear extract from +/+LDA11 cells treated with estradiol (10 nM) and TNF ⁇ and IL-1 (InM each for 40 min) as indicated were incubated with the -82 to -47 probe in the absence or presence of a 100-fold molar excess of oligonucleotides corresponding to the regions of -82 to -47 and -172 to -131 of the human IL-6 promoter or the vitellogenin ERE. Complexes formed were analyzed by EMSA.
  • Fig. 2c shows that when binding of the extracts to the oligonucleotide covering the NF-IL6 site (-172 to - 131) was investigated, several complexes were detected.
  • Nuclear extract from +/+LDA11 cells treated with estradiol (10 nM) and TNF ⁇ and IL-1 (InM each for 40 min) as indicated were incubated with the -172 to -131 probe in the absence or presence of a 100-fold molar excess the unlabeled oligonucleotide.
  • Complexes formed were analyzed by EMSA.
  • the arrows in Fig. 2c indicate the complexes A, B, and C formed upon induction with TNF ⁇ and IL-1.
  • +/+LDA11 cells were pretreated with cycloheximide (CHX) or the kinase inhibitor H7 for 5 min or with estradiol (10 nM) and/or ICI 164,384 (100 nM) for 24 h or 60 min before induction with TNF ⁇ and IL-1 (InM each for 30 min) .
  • Treatment was stopped by cell lysis and nuclear extracts were analyzed by EMSA using the -82 to -47 fragment as probe.
  • Arrows indicate the induced complexes A, B, and C
  • methylation interference experiments were carried out (Fig. 4) .
  • Nuclear +/+LDA11 extracts from cells induced with TNF ⁇ and IL-1 (InM each) were incubated with -82 to -47 probe that had been labeled either on the upper or the lower strand and subjected to limited DMS-methylation.
  • DNA from complexes A, B, and C and from the unretarded probe (F) was isolated, cleaved with piperidine, and electrophoresed on a 12% denaturing gel.
  • c-rel or an i munologically related factor is a component of a larger complex that binds the NF/cB site in the IL-6 promoter and functions as a constitutive repressor.
  • +/+LDA11 cells we could not detect any c-rel specific binding activity.
  • a number of other NF/cB unrelated proteins have been shown to bind to NFcB consensus sites. Those include aA-CRYBPl (Nakamura, T. , D.M. Donovan, K. Hamada, CM. Sax, B. Norman, J.R. Flanagan, K. Ozato, H. Westphal, and J.
  • estradiol induced complex formation with an NF/cB element (Shyamala, G. and M.C Guiot (1992) "Activation of kappa B-specific proteins by estradiol, " Proc. Natl. Acad. Sci. USA 89:10628-10632) .
  • the induced complex did not contain p50 or p65 and therefore may represent other factors.
  • proteins in complexes B and C are only immunologically related to p50 and p65 but actually of smaller size. Speculations that the p50 homologue p49 is part of the induced complexes and is responsible for the faster migration could not be confirmed. Although purified p49 bound the -82 to -47 IL-6 fragment and strongly cross-reacted with the anti- p50 antibody, the complex formed migrated even more slowly than the p50 complex. This corresponds to results obtained with other NFcB binding sites (Duckett, C.S., N.D. Perkins, T.F. Kowalik, R.M. Schmid, E.S. Huang, A.S. Baldwin, Jr., and G.J.
  • the finding that the anti-p50 antibody strongly cross-reacts with p49 indicates that the antibodies used may detect other NF/cB related proteins in the complexes.
  • the migration of complexes B and C could be higher than the migration observed with recombinant p50 due to conformational differences resulting from post-translatio-nal modification. This would correlate with the observation that the inclusion of anti-p50, abolishing complexes B and C, produced a supershifted complex (SI) migrating more slowly than the supershifted complex obtained with recombinant p50 (Fig. 5b, lanes 6-8) .
  • band A induced by IL-1 and TNF ⁇ in the absence of estradiol is composed of two different unresolved complexes, one (Al) that is also induced in the presence of estradiol and does not contain p50, and another complex (A2) containing p50 (or an immunologically related protein) .
  • Treatment with estradiol may increase the intensity of complex C at the expense of complex A2. If complex A2 represents a transcriptionally more active state, this could explain the inhibitory effect of estradiol on IL-6 expression.
  • TBP or TFIIDt directly interacts with NF/cB (Kerr, L.D., L.J. Ransone, P.
  • This particular protein is the NF/cB component containing the transactivation domain (Schmitz, M.L. and P.A. Baeuerle (1991) "The p65 subunit is responsible for the strong transcription activating potential of NF-kappa B," EMBO J. 10:3805-3817) .
  • the transactivation function may be differentially active.
  • the antibody shift experiments suggest that estradiol diminishes the A2 complex while increasing complex C
  • the slow migrating A2 complex may contain other factor(s) involved in the transactivation process.
  • the TATA-binding protein TBP, part of the TFIID complex has been reported to interact strongly with c-rel and p65, but not with p50 or p49 (Kerr, L.D.
  • Example 5 Efficacy-testing of Putative Cytokine Modulators Methods for testing the efficacy of putative cytokine modulators are provided. Each candidate compound is tested for its efficacy in modulating cytokine expression in cell lines, in animal models, and in controlled clinical studies using methods known to those skilled in the art and approved by the Food and
  • Drug Administration such as, but not limited to, those promulgated in The Federal Register 4_7 (no. 56) : 12558- 12564, March 23, 1982.
  • Methods are provided for determining whether an agent active in any of the methods listed above has little or no effect on healthy cells. Such agents are then formulated in a pharmaceutically acceptable buffer or in buffers useful for standard animal tests.
  • pharmaceutically acceptable buffer any buffer which can be used in a pharmaceutical composition prepared for storage and subsequent adminis ⁇ tration, which comprise a pharmaceutically effective amount of an agent as described herein in a pharmaceuti ⁇ cally acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) .
  • Preserva ⁇ tives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid may be added as preservatives. Id. at 1449.
  • antioxidants and suspending agents may be used. Id.
  • Method 1 Agents identified as having cytokine modulating activity are assessed for toxicity to cultured human cells. This assessment is based on the ability of liv ⁇ ing cells to reduce 2, 3 , -bis [2-methoxy-4-nitro-5-sulpho- nylphenyl] -5- [ (phenylamino) carbonyl] -2H-tetrazolium hydroxide] otherwise referred to as XTT (Paull et al. , J. Heterocyl. Chem. 25:763-767 (1987) ; Weislow et al. , (1989), J. Natl. Cane. Inst. 81:577) .
  • Viable mammalian cells are capable of reductive cleavage of an N-N bond in the tetrazole ring of XTT to form XTT formazan. Dead cells or cells with impaired energy metabolism are inca ⁇ pable of this cleavage reaction. The extent of the cleavage is directly proportional to the number of liv- ing cells tested.
  • Cells from a human cell line such as HeLa cells are seeded at 10 3 per well in 0.1 ml of cell culture medium (Dulbecco' s modified minimal essential medium supplemented with 10% fetal calf serum) in the wells of a 96 well microtiter plate.
  • Cytokine modulators are tested for cytotoxic effects on cultured human cell lines using incorporation of 35 S methionine into protein as an indicator of cell viability.
  • HeLa cells are grown in 96 well plates in Dulbecco' s minimal essential medium supplemented with 10% fetal calf serum and 50 ⁇ g/ml penicillin and streptomycin. Cells are initially seeded at 10 3 cells/well, 0.1 ml/well. Cells are grown for 48 hrs without exposure to the cytokine modulator, then medium is removed and varying dilutions of the cytokine modulator prepared in complete medium are added to each well, with control wells receiving no cytokine modulator.
  • Cells are incubated for an additional 48-72 hrs. Medium is changed every 24 hrs and replaced with fresh medium containing the same concentration of the cytokine modulators. Medium is then removed and replaced with complete medium without antifungal. Cells are incubated for 24 hr in the absence of cytokine modulator, then viability is estimated by the incor ⁇ poration of 35 S into protein. Medium is removed, re ⁇ placed with complete medium without methionine, and incubated for 30 min. Medium is again removed, and replaced with complete medium without methionine but containing 0.1 ⁇ Ci/ml 35 S methionine. Cells are incubat ⁇ ed for 3 hrs.
  • the invention features novel cytokine modulators discovered by the methods described above. It also includes novel pharmaceutical compositions which include cytokine modulators discovered as described above formu ⁇ lated in pharmaceutically acceptable formulations. Furthermore, the invention features a method for treating a subject inflicted with a pathological condition affected by the level of a cytokine by administering to that subject a therapeutically effective amount of a cytokine modulator. Such ad ⁇ ministration can be by any method known to those skilled in the art, for example, by topical application or by systemic administration.
  • terapéuticaally effective amount is meant an amount that relieves (to some extent) one or more symptoms of the disease or condition in the patient. Additionally, by “therapeutically effective amount” is meant an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with or causative of a mycotic disease or condition. Generally, it is an amount between about 1 nmole and 1 ⁇ mole of the molecule, dependent on its EC S0 and on the age, size, and disease associated with the patient. Other embodiments of this invention are disclosed in the following claims.

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Abstract

La présente invention a pour objet un procédé de triage pour détecter des agents de traitement de maladies et d'états pathologiques affectés par les cytokines. Ces agents interagissent directement ou indirectement avec un récepteur intracellulaire, qui, à son tour, module la liaison d'une protéine de type rel, d'un complexe de protéines de type rel, ou d'autres protéines de transcription sur un site rel sur le promoteur d'un gène de cytokine. Le récepteur intracellulaire peut être le récepteur d'÷strogène, les récepteurs d'acide rétinoïde, les récepteurs de rétinoïde X, le récepteur de glucocorticoïde, les récepteurs de progestérone, le récepteur d'androgène, les récepteurs d'hormone thyroïdienne, ou le récepteur de vitamine D. Les agents de sélection peuvent être utilisés pour traiter l'ostéoporose, la polyarthrite rhumatoïde, l'inflammation, le psoriasis, le sarcome de Kaposi, le choc septique et les myélomes multiples.
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US6692928B1 (en) 1996-07-19 2004-02-17 New England Medical Center Hospitals, Inc. Methods for identifying cardiovascular agents
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AU7377196A (en) * 1996-09-27 1998-04-17 Jim Cai Steroid receptor binding compositions and steroid-sparing preparations thereof in immunosuppression and inflammation
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EP1158297A4 (fr) * 1999-02-18 2002-04-10 Chugai Pharmaceutical Co Ltd Procede permettant l'identification d'un composant presentant une affinite pour le recepteur de la vitamine d
WO2001094951A2 (fr) * 2000-06-08 2001-12-13 Board Of Regents, The University Of Texas System Inhibiteurs d'inflammation induite par la proteine c reactive
KR20040026680A (ko) 2001-07-09 2004-03-31 콤비네이토릭스, 인코포레이티드 염증 질환 치료용 조합
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WO2005090282A1 (fr) 2004-03-12 2005-09-29 Ligand Pharmaceuticals Incorporated Composes modulateurs de recepteur d'androgenes et procedes
EP2511382A1 (fr) * 2011-04-14 2012-10-17 Université de Strasbourg Procédé pour identifier de nouvelles molécules anti-inflammatoires avec une transrepression directe réduite de gènes induite par glucocorticoïdes

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