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WO2003014745A2 - Procede d'identification de substances ayant une influence positive sur les affections inflammatoires - Google Patents

Procede d'identification de substances ayant une influence positive sur les affections inflammatoires Download PDF

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Publication number
WO2003014745A2
WO2003014745A2 PCT/EP2002/007937 EP0207937W WO03014745A2 WO 2003014745 A2 WO2003014745 A2 WO 2003014745A2 EP 0207937 W EP0207937 W EP 0207937W WO 03014745 A2 WO03014745 A2 WO 03014745A2
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Prior art keywords
protein
nhr
macrophage
substance
activator
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PCT/EP2002/007937
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English (en)
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WO2003014745A3 (fr
Inventor
Birgit Jung
Norbert Kraut
Stefan Müller
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Boehringer Ingelheim Pharma Gmbh & Co. Kg
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Application filed by Boehringer Ingelheim Pharma Gmbh & Co. Kg filed Critical Boehringer Ingelheim Pharma Gmbh & Co. Kg
Priority to EP02754896A priority Critical patent/EP1425587A2/fr
Priority to JP2003519426A priority patent/JP2005503787A/ja
Priority to MXPA04001128A priority patent/MXPA04001128A/es
Priority to CA002453913A priority patent/CA2453913A1/fr
Priority to AU2002321229A priority patent/AU2002321229A1/en
Publication of WO2003014745A2 publication Critical patent/WO2003014745A2/fr
Publication of WO2003014745A3 publication Critical patent/WO2003014745A3/fr

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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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors

Definitions

  • the present invention belongs to the field of modulation of inflammatory processes, in particular of chronic inflammatory airway diseases, in which macrophages play an important role.
  • the inflammatory processes can be modulated according to the invention by influencing the biological activity of a nuclear hormone receptor protein, which is identified to be involved in the inflammatory process.
  • CB chronic bronchitis
  • CB may occur with or without airflow limitation and includes chronic obstructive pulmonary disease (COPD).
  • COPD chronic obstructive pulmonary disease
  • CB is a complex disease encompassing symptoms of several disorders: chronic bronchitis which is characterized by cough and mucus hypersecretion, small airway disease, including inflammation and peribronchial fibrosis, emphysema, and airflow limitation.
  • CB is characterized by an accelerated and irreversible decline of lung function.
  • the major risk factor for developing CB is continuous cigarette smoking. Since only about 20% of all smokers are inflicted with CB, a genetic predisposition is also likely to contribute to the disease.
  • the initial events in the early onset of CB are inflammatory, affecting small and large airways.
  • An irritation caused by cigarette smoking attracts macrophages and neutrophils the number of which is increased in the sputum of smokers.
  • Perpetual smoking leads to an ongoing inflammatory response in the lung by releasing mediators from macrophages, neutrophils and epithelial cells that recruit inflammatory cells to sites of the injury. So far there is no therapy available to reverse the course of CB.
  • Smoking cessation may reduce the decline of lung function.
  • macrophages involved in an inflammatory process particularly in a chronic inflammatory airway disease, more particularly in chronic bronchitis or COPD, show a pattern of differentially expressed nucleic acid sequence and protein expression which differs from the pattern of gene expression of macrophages from healthy donors or donors in an irritated state, which latter do contain macrophages in an activated state. Therefore, macrophages show different activation levels under different inflammatory conditions.
  • macrophages involved in an inflammatory process in COPD smokers show different gene expression pattern than macrophages from healthy smokers, indicating that in COPD smokers macrophages are in a different, hereinafter named "hyperactivated" state.
  • the present invention provides for the possibility to inhibit the hyperactivation or to reduce the hyperactive state of a macrophage by allowing the identification of substances which modulate a nuclear hormone receptor protein involved in the hyperactivation or maintaining the hyperactive state.
  • chronic inflammatory airway disease as used hereinafter includes, for example, Chronic Bronchitis (CB) and Chronic Obstructive Pulmonary Disease (COPD).
  • CB Chronic Bronchitis
  • COPD Chronic Obstructive Pulmonary Disease
  • the preferred meaning of the term “chronic inflammatory airway disease” is CB and COPD, the more preferred meaning is CB or COPD.
  • the invention is based on the identification of a nucleic acid sequence differentially expressed in a hyperactivated macrophage compared to a macrophage which is not hyperactivated.
  • a nucleic acid sequence encodes for a nuclear receptor protein, which is involved in the hyperactivation or maintaining the hyperactive state of a macrophage involved in an inflammatory process, preferably in a chronic inflammatory airway disease.
  • Such differentially expressed nucleic acid sequence or protein encoded by such nucleic acid sequence is in the following also named differentially expressed nucleic acid sequence or protein of the invention, respectively.
  • the present invention teaches a link between phenotypic changes in macrophages due to differentially expressed nucleic acid sequence and protein expression pattern and involvement of macrophages in inflammatory processes and, thus, provides a basis for a variety of applications.
  • the present invention provides a method and a test system for determining the expression level of a macrophage protein of the invention or differentially expressed nucleic acid sequence of the invention and thereby provides e.g. for methods for diagnosis or monitoring of inflammatory processes with involvement of hyperactivated macrophages in mammalian, preferably human beings, especially such beings suffering from an inflammatory process, preferably in a chronic inflammatory airway disease, more preferably in chronic bronchitis or COPD.
  • the invention also relates to a method for identifying a substance by means of a differentially expressed nucleic acid sequence or protein of the invention, which substance modulates, i.e. acts as an inhibitor or activator on the said differentially expressed nucleic acid sequence or protein of the invention and thereby positively influences chronic inflammatory processes by inhibition of the hyperactivation or reduction of the hyperactive state of macrophages, and thereby allows treatment of mammals, preferably human beings, suffering from a said disease.
  • the invention also relates to a method for selectively modulating such a differentially expressed nucleic acid sequence or protein of the invention in a macrophage comprising administering a substance determined to be a modulator of said protein or differentially expressed nucleic acid sequence.
  • the present invention includes the use of said substances for treating beings in need of a treatment for an inflammatory process.
  • a differentially expressed nucleic acid sequence of the invention in a first step is identified which has a different expression pattern in a hyperactivated macrophage compared to a macrophage which is not hyperactivated.
  • this description deals particularly with investigation of macrophages involved in COPD, however, equivalent results may be obtained with samples from subjects suffering from other chronic inflammatory airway diseases, e.g. other chronic bronchitis symptoms.
  • the investigation of the different expression pattern leads to the identification of a series of differentially expressed nucleic acid sequences expressed in dependency on the activation state of a macrophage involved in an inflammatory process, as exemplified in the Examples hereinbelow.
  • differentially expressed nucleic acid sequence of the invention is identified by comparative expression profiling experiments using a cell or cellular extract from a hyperactivated macrophage, i.e. for example from the site of inflammation in COPD and from the corresponding site of control being not suffering from said disease, however, suffering under the same irritating condition like cigarette smoke exposure.
  • the proteins are identified which are encoded by the differentially expressed nucleic acid sequences, i.e. proteins playing a role in mediating the hyperactivation or in maintaining the hyperactivated state.
  • a class of differentially expressed nucleic acid sequences of the invention can be identified to encode a class of proteins which act as nuclear receptor protein of the invention which is characterized in that it is expressed in a macrophage that is hyperactivated according the invention at a lower or higher level than the control level in a macrophage which is not hyperactivated.
  • NHR-protein nuclear hormone receptor protein
  • NHR-protein is estrogen-related receptor ⁇ (ERR , SEQ ID NO. 1 and 7) or nuclear receptor subfamily 4 group A member 1 (NR4A1 , SEQ ID NO. 2 and 8), depicted in the sequence listing.
  • ERP estrogen-related receptor ⁇
  • NR4A1 nuclear receptor subfamily 4 group A member 1
  • the biological activity of a NHR-protein according to the present invention is dependent, for example, on recognition of and /or binding to a responsive DNA element influencing transcription of a DNA connected with said DNA element (e.g. ERR ⁇ responsive element (ERRE) or steroidogenic factor responsive element (SFRE)) or for example on recognition of and /or binding to an other protein resulting in a rendered transcription activity.
  • a responsive DNA element influencing transcription of a DNA connected with said DNA element e.g. ERR ⁇ responsive element (ERRE) or steroidogenic factor responsive element (SFRE)
  • the biological activity of a NHR-protein according to the invention is not limited to influence transcription activity.
  • the biological activity of a NHR-protein according to the invention for example facultatively comprises the regulation of apoptosis through a mechanism independent of transcription activity.
  • the invention also concerns functional equivalents, derivatives, variants, mutants and fragments of a NHR-protein, preferentially of the preferred proteins mentioned hereinbefore.
  • Functional in this context means having a function of the respective corresponding NHR-protein which is involved in its biological activity, e.g. DNA and /or protein recognition.
  • the biological activity of a NHR-protein expressed at a lower level than the control level is preferably activated in order to inhibit hyperactivation or reduce a hyperactivated state of a macrophage, whereby the biological activity of a NHR-protein which is expressed at a higher level than the control level is preferably inhibited in order to inhibit hyperactivation or reduce a hyperactivated state of a macrophage.
  • the present invention concerns a test method for determining whether a substance is an activator or inhibitor of a NHR-protein. Since a NHR- protein is involved in a chronic inflammatory airway disease and plays a role in mediating inflammation, a substance modulating the biological activity of a NHR- protein can be used for treating a chronic inflammatory airway diseases or can be used as lead compound for optimization of the function of the substance in a way that the optimized substance is suitable for treating chronic inflammatory airway diseases.
  • a method for determining whether a substance is an activator or an inhibitor of a function of a NHR-protein deregulated in a hyperactivated macrophage can be characterized in that the method comprises contacting the NHR-protein or variant, mutant or fragment thereof having a NHR-protein function with a substance to be tested whether it is an inhibitor or activator of a desired function of the NHR-protein, and measuring whether the desired function is inhibited or activated.
  • the desired function can be the biological activity of a NHR-protein of the invention.
  • Said measuring can be performed directly e.g. with well known procedures allowing to measure direct binding of a said protein with a said substance, or indirectly, e.g. using well known reporter systems allowing to draw conclusions about the binding of a said protein with a said substance.
  • test system for performing a method of the invention, a test system according to the invention can be used.
  • the present invention also concerns a test system for determining whether a substance is an activator or an inhibitor of a NHR-protein function.
  • a test system useful for performing a method of the invention comprises a cellular or a cell-free system.
  • one embodiment of the invention concerns a test system that is designed in a way to allow the testing of substances acting on the expression level of the differentially expressed nucleic acid sequence e.g. using expression of a reporter-gene, e.g. luciferase gene or the like, as a measurable readout.
  • Another embodiment of the invention concerns a test system that is designed in a way to allow the testing of substances directly interacting with a function, e.g. the recognition and/or binding activity, of the NHR-protein or interfering with the activation of a function of the NHR-protein by a natural or an artificial but appropriate activator of the NHR-protein, e.g. an appropriate ligand.
  • a test system of the invention comprises, for example, elements well known in the art.
  • cell-free systems may include, for example, a NHR-protein or a functional equivalent, derivative, variant, mutant or fragment of a NHR-protein, a nucleic acid encoding a NHR-protein or encoding a functional equivalent, derivative, variant, mutant or fragment of a NHR-protein in soluble or bound form or in cellular compartments or vesicles.
  • Suitable cellular systems include, for example, a suitable prokaryotic cell or eukaryotic cell, e.g.
  • a cell suitable for use in a said test system of the invention may be obtained by recombinant techniques, e.g. after transformation or transfection with a recombinant vector suitable for expression of a desired NHR- protein or functional equivalent, derivative, variant, mutant or fragment of a NHR- protein, or may e.g. be a ceil line or a cell isolated from a natural source expressing a desired NHR-protein or functional equivalent, derivative, variant, mutant or fragment of NHR-protein.
  • a test system of the invention may include a natural or artificial ligand of a NHR-protein if desirable or necessary for testing whether a substance of interest is an inhibitor or activator of a NHR-protein.
  • a test method comprises measuring a read-out, e.g. a phenotypic change in the test system, for example, if a cellular system is used a phenotypic change of the cell is monitored.
  • a read-out e.g. a phenotypic change in the test system
  • Such change may be a change in a naturally occurring or artificial response, e.g. a reporter gene expression of the cell to NHR-protein activation or inhibition, e.g. as detailed in the Examples hereinbelow.
  • a test method according to the invention can on the one hand be useful for high throughput testing suitable for determining whether a substance is an inhibitor or activator of the invention, but also e.g. for secondary testing or validation of a hit or lead substance identified in high throughput testing.
  • the present invention also concerns a substance identified in a method according to the invention to be an inhibitor or activator of a NHR-protein.
  • a substance of the present invention is any compound which is capable of modulating preferably activating or inhibiting a function of a NHR-protein according to the invention.
  • An example of a way to activate or inhibit a function of a NHR-protein is by influencing the expression level of said NHR-protein.
  • Another example of a way to activate or inhibit a function of a NHR-protein is to apply a substance directly binding the NHR- protein and thereby activating or blocking functional domains of said NHR-protein, which can be done reversibly or irreversibly, depending on the nature of the substance applied.
  • a substance useful for activating or inhibiting biological activity of a NHR-protein includes a substance acting on the expression of differentially expressed nucleic acid sequence, for example a nucleic acid fragment hybridizing with the corresponding gene or regulatory sequence and thereby influencing gene expression.
  • the invention concerns, for example, a substance which is a nucleic acid sequence coding for the gene of a NHR-protein, or a fragment, derivative, mutant or variant of such a nucleic acid sequence, which nucleic acid sequence or a fragment, derivative, mutant or variant thereof is capable of influencing the gene expression level, e.g. a nucleic acid molecule suitable as antisense nucleic acid, ribozyme, or for triple helix formation.
  • the invention also concerns a substance which is e.g. an antibody or an organic or inorganic compound directly binding to or interfering with the activation of a NHR- protein or directly binding to a NHR-protein and thereby affecting its biological activity.
  • a substance which is e.g. an antibody or an organic or inorganic compound directly binding to or interfering with the activation of a NHR- protein or directly binding to a NHR-protein and thereby affecting its biological activity.
  • the present invention relates to a method for determining an expression level of a NHR-protein by determining the level of a nucleic acid coding for a NHR-protein, more preferably determining the level of respective messenger RNA, or determining the level of a NHR-protein itself, in a cell, preferably in a macrophage, more preferably in a macrophage isolated form a site of inflammation, even more preferably from a site of inflammation in a subject suffering from a chronic inflammatory airway disease.
  • Such a method can be used, for example, for testing whether a substance is capable of influencing differentially expressed nucleic acid sequence expression levels in a method outlined above for determining whether a substance is an activator or inhibitor according to the present invention.
  • a method for determining an expression level according to the invention can, however, also be used for testing the activation state of a macrophage, e.g. for diagnostic purposes or for investigation of the success of treatment for a disease which is caused by the hyperactivated macrophage, e.g. for monitoring.
  • Said macrophage is preferably a mammalian, more preferably a human cell.
  • macrophages of the present invention are preferably obtainable from the site of inflammation in a mammal and more preferably from a site of inflammation in a human being.
  • the invention also relates to a method for diagnosis of a chronic inflammatory disease, or monitoring of such disease, e.g. monitoring success in treating beings in need of treatment for such disease, comprising determining an expression level of a nucleic acid coding for a a NHR-protein, preferably messenger RNA, or a a NHR-protein itself in a macrophage.
  • the present invention also relates to the use of a substance according to the invention for the treatment for a chronic inflammatory airway disease.
  • a pharmaceutical composition comprising at least one of the substances according to the invention determined to be an activator or an inhibitor.
  • the composition may be manufactured in a manner that is itself known, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, powdering, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the substances can be tested in animal models for example an animal suffering from an inflammatory airway disorder or a transgenic animal expressing a NHR-protein according to the invention.
  • Toxicity and therapeutic efficacy of a substance according to the invention can be determined by standard pharmaceutical procedures, which include conducting cell culture and animal experiments to determine the IC 50 , LD 50 and ED 50 .
  • the data obtained are used for estimating the animal or more preferred the human dose range, which will also depend on the dosage form (tablets, capsules, aerosol sprays ampules, etc.) and the administration route (for example transdermal, oral, buccal, nasal, enteral, parenteral, inhalative, intratracheal, or rectal).
  • a pharmaceutical composition containing at least one substance according to the invention as an active ingredient can be formulated in conventional manner. Methods for making such formulations can be found in manuals, e.g. "Remington Pharmaceutical Science”. Examples for ingredients that are useful for formulating at least one substance according to the present invention are also found in WO 99/18193, which is hereby incorporated by reference.
  • the invention concerns a method for treating a chronic inflammatory airway disease according to the invention.
  • Such method comprises administering to a being, preferably to a human being, in need of such treatment a suitable amount of a pharmaceutical composition comprising at least one substance determined to be an activator or inhibitor by a method according to the invention for determining whether a substance is an activator or an inhibitor of a NHR-protein according to the invention.
  • the invention relates to a method for selectively modulating NHR-protein concentration in a macrophage, comprising administering a substance determined to be an activator or inhibitor of a NHR-protein according to the invention.
  • BAL is filtered through sterile gauze to remove debris.
  • the cells are washedrtwice in HBSS, resuspended in 1 ml HBSS (Hank's Balanced Salt Solution) and counted.
  • the macrophages are spun to a pellet using 15 ml Falcon blue-cap polypropylen, resuspended in Trizol reagent (Gibco BRL Life Technologies) at a concentration of 1 ml Trizol reagent per 10 million cells and then frozen at -70°C.
  • RNA is extracted from macrophage samples obtained according to Example 1.3.
  • Cell suspensions in Trizol are homogenized through pipetting and incubated at room temperature for 5 minutes.
  • 200 ⁇ l chloroform per ml Trizol is added, the mixture carefully mixed for 15 seconds and incubated for 3 more minutes at room temperature.
  • the samples are spun at 10000g for 15 minutes at 4°C.
  • the upper phase is transferred into a new reaction tube and the RNA is precipitated by adding 0.5 ml isopropanol per ml Trizol for 10 minutes at room temperature.
  • the precipitate is pelleted by using a microcentifuge for 10 minutes at 4°C with 10000g, the pellet is washed twice with 75% ethanol, air dried and resuspended in DEPC- H 2 O.
  • RNA cleanup with Qiagen RNeasy Total RNA isolation kit is performed in order to improve the purity of the RNA.
  • the purity of the RNA is determined by agarose gelelectrophoresis and the concentration is measured by UV absorption at 260 nm.
  • RNA 5 ⁇ g of each RNA is used for cDNA synthesis.
  • First and second strand synthesis are performed with the Superscript Choice system (Gibco BRL Life Technologies).
  • T7-(dt) 24 primer (sequence: ggccagtgaa ttgtaatacg actcactata gggaggcggt ttttttttttttttttttttttttttttttttttttttttttttttttttt, SEQ ID NO. 9) are heated up to 70°C for 10 minutes and then cooled down on ice for 2 minutes.
  • First strand buffer to a final concentration of 1x, DTT to a concentration of 10 mM and a dNTP mix to a final concentration of 0.5 mM are added to a total volume of 18 ⁇ l.
  • the reaction mix is incubated at 42°C for 2 minutes and 2 ⁇ l of Superscript II reverse transcriptase (200 U/ ⁇ l) are added.
  • For second strand synthesis 130 ⁇ l of a mix containing 1.15x second strand buffer, 230 ⁇ M dNTPs, 10 U E.coli DNA ligase (10U/ ⁇ l), E.coli DNA polymerase (10 U/ ⁇ l), RNase H (2U/ ⁇ l) is added to the reaction of the first strand synthesis and carefully mixed with a pipette.
  • Second strand synthesis is performed at 16°C for 2 hours, then 2 ⁇ l of T4 DNA polymerase (5 U/ ⁇ l) are added, incubated for 5 minutes at 16°C and the reaction is stopped by adding 10 ⁇ l 0.5 M EDTA.
  • the double stranded cDNA is purified.
  • the cDNA is mixed with an equal volume of phenol:chloroform:isoamylalcohol (25:24:1 ) and spun through the gel matrix of phase lock gels (Eppendorf) in a microcentrifuge in order to separate the cDNA from unbound nucleotides.
  • the aqueous phase is precipitated with ammoniumacetate and ethanol.
  • the cDNA is used for in vitro transcription.
  • cRNA synthesis is performed with the ENZO BioArray High Yield RNA Transcript Labeling Kit according to manufacturer's protocol (ENZO Diagnostics).
  • the cDNA is incubated with 1x HY reaction buffer, 1x biotin labeled ribonucleotides, 1x DTT, 1x RNase Inhibitor Mix and 1x T7 RNA Polymerase in a total volume of 40 ⁇ l for 5 hours at 37°C. Then, the reaction mix is purified via RNeasy columns (Qiagen), the cRNA precipitated with ammonium acetate and ethanol and finally resuspended in DEPC-treated water. The concentration is determined via UV spectrometry at 260 nm. The remaining cRNA is incubated with 1x fragmentation buffer (5x fragmentation buffer: 200 mM Tris acetate, pH 8.1 , 500 mM KOAc, 150 mM MgOAc) at 94°C for 35 minutes.
  • 1x fragmentation buffer 5x fragmentation buffer: 200 mM Tris acetate, pH 8.1 , 500 mM KOAc, 150 mM MgOAc
  • cRNA For hybridization of the DNA chip 15 ⁇ g of cRNA is used, mixed with 50 pM biotin- labeled control B2 oligonucleotide, sequence: gtcgtcaaga tgctaccgtt cagga (SEQ ID NO: 10), 1x cRNA cocktail, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated BSA, 1x MES (2-[N-morpholino]-ethanesulfonic acid) hybridization buffer in a total volume of 300 ⁇ l.
  • the hybridization mixture is heated up to 99°C for 5 minutes, cooled down to 45°C for 10 minutes and 200 ⁇ l of the mix are used to fill the probe array.
  • the hybridization is performed at 45°C at 60 rpm for 16 hours. After the hybridization the hybridization mix on the chip is replaced by 300 ⁇ l non- stringent wash buffer (100 mM MES, 100 mM NaCI, 0.01 % Tween 20). The chip is inserted into an Affymetrix Fluidics station and washing and staining is performed according to the EukGE-WS2 protocol.
  • the staining solution per chip- consists of 600 ⁇ l 1x stain buffer (100 mM MES, 1 M NaCI, 0.05% Tween 20), 2 mg/ml BSA, 10 ⁇ g/ml SAPE (streptavidin phycoerythrin) (Dianova), the antibody solution consists of 1x stain buffer, 2 mg/ml BSA, 0.1 mg/ml goat IgG, 3 ⁇ g/ml biotinylated antibody. After the washing and staining procedure the chips are scanned on the HP Gene
  • Data Analysis is performed by pairwise comparisons between chips hybridized with RNA isolated from COPD smokers and chips hybridized with RNA isolated from healthy smokers.
  • Estrogen-related receptor alpha is an orphan member of the superfamily of nuclear hormone receptors. It binds to a single consensus half-site of the ERR ⁇ response element (ERRE) and to the steroidogenic factor 1 response element (SFRE) (Vanacker et al. 1999). It is found that genes with these sites in their promoter region are targets for transcription activation by ERR ⁇ . These genes are for example medium-chain acyl coenzyme A dehydrogenase (MCAG), osteopontin, and the thyroid hormone receptor ⁇ (Vanacker et al. 1998). Due to the activation of MCAG it is assumed that ERR ⁇ is involved in regulating the energy balance in vivo (Sladek et al. 1997).
  • the ERR ⁇ (ace. L38487) is consistently found upregulated (42%) in COPD smokers compared to healthy smokers. This is shown "fold change” values (Table 1). The p values for two separate groups comparing COPD smokers and healthy smokers are 0.03 and 0.15. Tablel : Fold change values (FC) for comparisons between obstructed smoker and healthy smokers. On average is upregulated by 2.3fold, the median is 1.6fold.
  • ERR ⁇ is cloned from a total RNA extracted from human kidney. 5 ⁇ g RNA is reverse transcribed into cDNA with 5 ng oligo(dt) 18 primer, 1x first strand buffer, 10 mM DTT, 0.5 mM dNTPs and 2 U Superscript II (Gibco BRL) at 42°C for 50 minutes. Then, the reaction is terminated at 70°C for 15 minutes and the cDNA concentration is determined by UV-spectrophotometry.
  • ERR ⁇ 100 ng of the cDNA and 10 pmoles of sequence-specific primers for ERR ⁇ (forward primer: ggggacaagt ttgtacaaaa aagcaggcta tgggattgga gatgagctc; SEQ ID No. 3 and reverse primer: ggggaccact ttgtacaaga aagctgggtt cagtccatca tggcctcgag SEQ ID No. 4) are used for PCR.
  • Reaction conditions are: 2 minutes of 94°C, 35 cycles with 30 seconds at 94°C, 30 seconds at 53°C, 90 seconds at 72°C, followed by 7 minutes at 72°C with Taq DNA-polymerase.
  • the reaction mix is separated on a 2% agarose gel, a band of about 1000bp is cut out and purified with the QIAEX II extraction kit (Qiagen). The concentration of the purified band is determined and about 120 ng are incubated with
  • BP clonase reaction buffer 1x BP clonase reaction buffer, BP clonase enzyme mix in a total volume of 20 ⁇ l for 60 minutes at 25°C. Then, reactions are incubated with 2 ⁇ l of proteinase K and incubated for 10 minutes at 37°C. The reaction mix is then electroporated into competent DB3.1 cells and plated on Kanamycin-containing plates. Clones are verified by sequencing. A clone, designated pDONR-ERR ⁇ , with identical sequence to the database entry (ace. X51416) is used for further experiments.
  • the vector containing ERR ⁇ described under 1.1. is used to transfer the cDNA for
  • ERR ⁇ to the expression vector pcDNA3.1(+)/attR that contains the "attR1" and “attR2" recombination sites of the Gateway cloning system (Life Technologies) where ERR ⁇ is expressed under the control of the CMV promoter.
  • 150 ng of the "entry vector" pDONR-ERR ⁇ is mixed with 150 ng of the "destination vector" pcDNA3.1(+)/attR, 4 ⁇ l of the LR Clonase enzyme mix, 4 ⁇ l LR Clonase reaction buffer, added up with TE (Tris/EDTA) to 20 ⁇ l and incubated at 25°C for 60 minutes.
  • a colony that contains pcDNA3.1 (+)/attR with ERR ⁇ as an insert is designated pcDNA/ERR ⁇ and used for transfection studies.
  • ERR ⁇ Mon ⁇ cytic cell lines are seeded in a 35 mm petri dish and cultivated in RPMI 1640 media containing 10% FCS supplemented with 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 2 mM glutamine, and 1x non-essential amino acids over night. Cells that are grown to a confluency of 50-80% are used for transfection. 6 ⁇ l FuGene ⁇ (Roche Biochemicals) is added to 100 ⁇ l of culture media without serum and equilibrated for 5 minutes at room temperature. Then, 2 ⁇ g of purified pcDNA/ERR ⁇ is added to the prediluted FuGene ⁇ solution, gently mixed, and further incubated at 5 room temperature for 15 minutes.
  • the media is aspirated from the cells and 4 ml of fresh media is added to the cells.
  • the FuGene6/DNA solution is added dropwise to the cells and distributed evenly by swirling of the media.
  • the media is aspirated and replaced by RPMI 1640, 10% FCS, 2 mM glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 200 ⁇ g/ml G418.
  • the media is replaced daily until dead cells and debris is washed out until single colonies of cells are visible. Single colonies are isolated by separation with cloning cylinders and releasing them from the surface by addition of 100 ⁇ l of 1x trypsin/EDTA.
  • Cells are transferred from the cloning cylinders to 4 ml of media and plated in 6 well-plates. Single clones are expanded and the expression of ERR ⁇ in 5 several clones is tested via Western blotting. A cell clone with the highest expression of ERR ⁇ is used for further studies.
  • vectors allow the expression of recombinant his-tagged ERR ⁇ in bacteria under the control of the T7 promoter.
  • 150 ng of the "entry vector” pDONR-ERR ⁇ is 5 mixed with 150 ng of the "destination vector” gpET28abc/attR, 4 ⁇ l of the LR Clonase enzyme mix, 4 ⁇ l LR Clonase reaction buffer, added up with TE (Tris/EDTA) to 20 ⁇ l ,.. handled and incubated at 25°C for 60 minutes. Then, 2 ⁇ l of proteinase K solution is added and incubated for 10 minutes at 37°C.
  • TE Tris/EDTA
  • 1 - ⁇ l of the reaction mix is transformed into 50 ⁇ l DH5 ⁇ by a heat-shock of 30 seconds at 42°C after incubating cells with DNA for 0 30 minutes on ice. After heat-shock of the cells 450 ⁇ l of S.O.C. is added and cells are incubated at 37°C for 60 minutes. Cells (100 ⁇ l) are plated on LB plates containing 100 ⁇ g/ml ampicillin and incubated over night. A colony that contains gpET28abc/attR with ERR ⁇ fused to the his-tag in the correct reading frame is designated gpET/ERR ⁇ and used for expression of ERR ⁇ in bacteria.
  • 1 I LB broth including 100 ⁇ g/ml ampicillin is inoculated with 0.5 ml of an overnight culture of E. coli M15(pREP4) that carries pDONR-ERR ⁇ .
  • the culture is incubated at 37°C with vigorous shaking until OD 600 of 0.6.
  • Expression is induced by adding 1 mM IPTG and the culture is grown further for 4 hours.
  • Cells are harvested by centrifugation at 4000xg for 20 minutes at 4°C. Pellet is frozen at -20°C.
  • lysis buffer 50 mM NaH 2 PO4, pH 8.0, 300 mM NaCI, 10 mM imidazole. Then, lysozyme is added to 1 mg/ml and incubated on ice for 30 minutes. Then, cells are sonicated (six bursts of 10 seconds at 300 W). 10 ⁇ g/ml RNase A and 5 ⁇ g/ml DNase I is added and incubated on ice for 10 minutes. Then, lysates are cleared by spinning debris at 10000xg for 20 minutes at 4°C.
  • protease inhibitors 40 ⁇ g/ml bacitracin, 4 ⁇ g/ml leupeptin, 4 ⁇ g/ml chymostatin, 10 ⁇ g/ml pefabloc, 100 ⁇ M PMSF
  • protease inhibitors 40 ⁇ g/ml bacitracin, 4 ⁇ g/ml leupeptin, 4 ⁇ g/ml chymostatin, 10 ⁇ g/ml pefabloc, 100 ⁇ M PMSF
  • 3 ml of Ni-NTA resin Qiagen
  • Binding to the resin is allowed for 60 minutes at 4°C during gentle shaking.
  • the fluorescence polarisation assay is used in order to find substances that directly inhibit the interaction of the nuclear hormone receptor with its DNA binding site:Two complementary oligos containing the binding site (AGGTCA) for ERR ⁇ are synthesized.
  • the complementary oligo cgggtagagg tcacagtgac ctctacccgt (SEQ ID No.6) is synthesized without label.
  • 10 ⁇ M of the TAMRA-labeled oligo and 15 ⁇ M of the complementary oligo are mixed in 10 mM Tris/HCI, pH 7.5, 80 mM NaCI, 1 mM
  • Oligos are incubated at 95°C for 5 minutes (reaction tube in a 2 I beaker filled with boiling water) and cooled down to room temperature over night. DNA-binding assays are performed in 96-well Fluotrac 200 plates (Greiner). Per well 150 ⁇ l 20 nM of the annealed oligo are incubated with 40 nM of the nuclear hormone receptor in a reaction buffer containing 10 mM Tris/HCI, pH 7.5, 100 mM NaCI, 0.1 mM EDTA, 1%
  • Binding is allowed at 27°C for 2 hours. Substances according to the invention are added in a concentration range from 0.1 - 100 ng/ml. Fluorescence is monitored with a Polarion fluorometer (Tecan). Wells including binding buffer and oligo are used as controls. 1 nM fluorescein is used to calibrate the fluorometer.
  • Monocytic/macrophage cell lines are stimulated with various stimuli, like 10 nM PMA,
  • Stimulation of cells by cigarette smoke is performed by a smoke-enriched media.
  • 100 ml RPMI media without supplements is perfused with the cigarette smoke of 2 cigarettes.
  • the smoke of the cigarettes is pulled into a 50 ml syringe (about 20 volumes of a 50-ml volumes per cigarette) and then perfused into the media.
  • the pH of the media is adjusted to 7.4, and the media is filtersterilized through a 0.2 ⁇ m filter.
  • Cells are resuspended in smoke-enriched media and incubated for 10 minutes at 37°C at a density of 1x10 6 cells/ml. Then, cells are washed twice with RPMI 1640 and seeded in flasks or 24-well plates (MonoMac 6) for the times indicated above.
  • RNAs are isolated with the Qiagen RNeasy Total RNA Isolation Kit (Qiagen) according to the manufacturer's protocol. Purified RNA is used for TaqMan analysis. The expression levels of cytokines TNF ⁇ , IL-1 ⁇ , IL-8, and IL-6 are measured.
  • Proteins in the supernatants of the cultured and stimulated cells are precipitated by adding TCA to a final concentration of 10%. Precipitates are washed twice with 80% ethanol and pellets are resuspended in 50 mM Tris/HCI, pH 7.4, 10 mM MgCI 2 , 1 mM EDTA. Protein concentration is determined via the Bradford method and 50 ⁇ g of each sample are loaded on 12% SDS polyacrylamide gels. Gels are blotted onto PVDF-membranes, blocked for 1 hour in 5% BSA in TBST, and incubated for 1 hour with commercially available antibodies against human TNF ⁇ , IL-1 ⁇ , IL-8, and IL-6.
  • the procedure is identical to the one used for cytokines.
  • Antibodies used for Western blotting are against human MMP-1 , MMP-7, MMP-9, and MMP-12.
  • Protease activity is determined with a fluorescent substrate.
  • Supernatants isolated from stimulated and unstimulated cells are incubated in a total volume of 50 ⁇ l with 1 ⁇ M of the substrate (Dabcyl-Gaba-Pro-Gln-Gly-Leu-Glu
  • EDANS EDANS-Ala-Lys-NH2 (Novabiochem)
  • Positive controls are performed with 125 ng purified MMP-12 per reaction.
  • Protease activity is determined by fluorometry with an excitation at 320 nm and an emission at 405 nm.
  • a chemotaxis (Boyden) chamber In an alternative assay to determine proteolytic activity and cell migration a chemotaxis (Boyden) chamber is used.
  • cells 10 5 cells per well
  • cells In the lower compartment chemoattractants like leukotriene B 4 (10 ng/ml), MCP-1 (10 ng/ml) are added to the media.
  • chemoattractants like leukotriene B 4 (10 ng/ml)
  • MCP-1 (10 ng/ml) are added to the media.
  • cells on the undersurface that have traversed the Matrigel are fixed with methanol, stained with the Diff-Quik staining kit (Dade Behring) and counted in three high power fields (400x) by light microscopy.
  • Diff-Quik staining kit Diff-Quik staining kit
  • chemotaxis In order to determine chemotaxis a 48 well chemotaxis (Boyden) chamber (Neuroprobe) is used. Cells are starved for 24 hours in RPMI media without FCS. Chemoattractants, (50 ng/ml IL-8 , 10 ng/ml MCP-1 , 10 nM lipoxin A4, leukotriene B 4 (10 ng/ml), MCP-1 (10 ng/ml) and substances according to the invention are diluted in RPMI media without FCS and 30 ⁇ l is placed in the wells of the lower compartment. The upper compartment is separated from the lower compartment by a polycarbonate filter (pore size 8 ⁇ m).
  • a polycarbonate filter pore size 8 ⁇ m
  • Cells are harvested, washed in PBS and resuspended (4x10 6 /ml) in PBS and 1 ⁇ M BCECF ((2'-7'-bis-(carboxethyl)-5(6')-carboxyfluorescein acetoxymethyl) ester (Calbiochem), and incubated for 20 minutes at 37°C.
  • Cells are washed in PBS and resuspended (3.3x10 6 /ml) in PBS containing 0.1% BSA.
  • 3x10 5 cells (90 ⁇ l) are added to each well of a 96-well flat bottom plate coated with laminin (Becton Dickinson) and allowed to settle for 10 minutes.
  • Substances according to the invention are added and plates are incubated for 20 minutes at 37°C. Cells are washed with PBS containing 0.1% BSA and adherent cells are solubilized with 100 ⁇ l of 0.025 M NaOH and 0.1% SDS. Quantification is performed by fluorescence measurement.
  • 40 ⁇ l of a. dispersed suspension of heat-inactivated Saccharomyces boulardii (20 yeast/cell) are added to each well. Cells are incubated for three more hours, washed twice with PBS and cytocentrifuged. The cytospin preparations are stained with May-Gr ⁇ nwald-Giemsa and phagocytosed particles are counted by light microsopy.
  • cells are seeded in a capsule of the cytosensor in RPMI 1640, 2.5% FCS and grown over night at 37°C in 5% CO 2 in a humidified atmosphere. Before use, cells are washed with serum-free RPMI 1640, 10 mM HEPES (pH 7.4). Substances according to the invention (0.1 - 100 ng/ml) are added at time zero. Inhibition of MCAD-mediated and ERR ⁇ -driven acidification of the medium is monitored over a period of 120 minutes with cells treated with serum-free RPM1 1640, 10 mM HEPES (pH 7.4) set as 100%.
  • NR4A1 nuclear receptor subfamily 4, group A, member 1
  • NR4A1 A gene that is identified as consistently downregulated in individuals with COPD codes for NR4A1 which is an orphan member of the nuclear hormone receptor superfamily of transcription factors. It mediates cell proliferation in response to growth factors in the nucleus. Besides, NR4A1 also regulates apoptosis through a mechanism independent of transcriptional activity. In response to apoptotic stimuli, NR4A1 is translocated from the nucleus to the cytoplasm, where it targets mitochondria to induce cytochrome c release and apoptosis (Li et al. 2000).
  • NR4A1 (ace. D49728) is consistently found downregulated (44%) in COPD smokers compared to healthy smokers. This is shown by "fold change” values (Table 2 ). The p values for comparing two groups of COPD smokers and healthy smokers are 0.15 and 0.009.
  • the protein is cloned and assays are performed in an analogous manner to the cloning and assays described hereinbefore.
  • Apoptosis Assay (especially for NR4A1 )
  • the assay to determine the number of apoptotic cells is performed with the terminal transferase kit by Roche Diagnostics (cat. No. 220582).
  • Cell lines stably expressing the nuclear hormone receptor are seeded in 8-well tissue culture plates with 5x10 4 per ml and stimulated with PMA (100ng/ml) to induce apoptosis.
  • substances according to the invention are added to the cells ranging from 1 to 1000 ng/ml. 3 to 6 hours after stimulation cells are washed with PBS, 1 mM MgCI 2 , fixed with 3% paraformaldehyde in PBS for 10 minutes and treated twice with PBS/50 mM NH 4 CI for 5 minutes. Then, cells are treated with for 5 minutes with PBS/0.5% Triton
  • reaction mix 200 ⁇ l reaction mix consists of 40 ⁇ l 5x reaction buffer, 20 ⁇ l 25 mM

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Abstract

L'invention concerne des protéines NHR impliquées dans des processus inflammatoires. L'invention concerne également la modulation de la fonction desdites protéines NHR, cette modulation permettant d'exercer une influence positive sur les maladies inflammatoires.
PCT/EP2002/007937 2001-08-06 2002-07-17 Procede d'identification de substances ayant une influence positive sur les affections inflammatoires WO2003014745A2 (fr)

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Application Number Priority Date Filing Date Title
EP02754896A EP1425587A2 (fr) 2001-08-06 2002-07-17 Procede d'identification de substances anti-inflammatoirs
JP2003519426A JP2005503787A (ja) 2001-08-06 2002-07-17 炎症症状にポジティブに影響を与える物質の特定方法
MXPA04001128A MXPA04001128A (es) 2001-08-06 2002-07-17 Metodo para identificar sustancias que afectan realmente condiciones inflamatorias.
CA002453913A CA2453913A1 (fr) 2001-08-06 2002-07-17 Procede d'identification de substances ayant une influence positive sur les affections inflammatoires
AU2002321229A AU2002321229A1 (en) 2001-08-06 2002-07-17 Method for identifying anti-inflammatory drugs

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005074969A2 (fr) * 2004-02-07 2005-08-18 Bayer Healthcare Ag Diagnostics et traitements de maladies associees au recepteur nucleaire humain nr4a1 (nr4a1)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999024471A1 (fr) * 1997-11-10 1999-05-20 The Research Foundation Of State University Of New York Recepteur d'opiaces, de cannabinoides et d'oestrogenes
WO2000053563A1 (fr) * 1999-03-11 2000-09-14 Nuclear Receptor Research Limited Nouveaux ligands de recepteurs nucleaires ppar
WO2001022988A1 (fr) * 1999-09-30 2001-04-05 Aubin Jane E Recepteur associe a l'oestrogene, err alpha, regulateur de la formation osseuse
WO2002052270A2 (fr) * 2000-12-22 2002-07-04 Boehringer Ingelheim Pharma Gmbh & Co. Kg Methode permettant d'identifier des substances qui influencent positivement des affections inflammatoires de maladies chroniques inflammatoires des voies respiratoires
WO2002052036A2 (fr) * 2000-12-22 2002-07-04 Boehringer Ingelheim Pharma Gmbh & Co. Kg Procede d'identification de substances ayant une influence positive sur les conditions inflammatoires des maladies inflammatoires chroniques des voies aeriennes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999024471A1 (fr) * 1997-11-10 1999-05-20 The Research Foundation Of State University Of New York Recepteur d'opiaces, de cannabinoides et d'oestrogenes
WO2000053563A1 (fr) * 1999-03-11 2000-09-14 Nuclear Receptor Research Limited Nouveaux ligands de recepteurs nucleaires ppar
WO2001022988A1 (fr) * 1999-09-30 2001-04-05 Aubin Jane E Recepteur associe a l'oestrogene, err alpha, regulateur de la formation osseuse
WO2002052270A2 (fr) * 2000-12-22 2002-07-04 Boehringer Ingelheim Pharma Gmbh & Co. Kg Methode permettant d'identifier des substances qui influencent positivement des affections inflammatoires de maladies chroniques inflammatoires des voies respiratoires
WO2002052036A2 (fr) * 2000-12-22 2002-07-04 Boehringer Ingelheim Pharma Gmbh & Co. Kg Procede d'identification de substances ayant une influence positive sur les conditions inflammatoires des maladies inflammatoires chroniques des voies aeriennes

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Title
NUCLEAR RECEPTORS NOMENCLATURE COMMITTEE, 1999: "A unified nomenclature system for the nuclear receptor superfamily." CELL, vol. 97, 16 April 1999 (1999-04-16), pages 161-163, XP002267664 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005074969A2 (fr) * 2004-02-07 2005-08-18 Bayer Healthcare Ag Diagnostics et traitements de maladies associees au recepteur nucleaire humain nr4a1 (nr4a1)
WO2005074969A3 (fr) * 2004-02-07 2005-10-20 Bayer Healthcare Ag Diagnostics et traitements de maladies associees au recepteur nucleaire humain nr4a1 (nr4a1)

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