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WO2001079539A2 - Procede d'utilisation de constructions transfectables composees de luciferase et du promoteur du gene periode 1 humain pour l'identification de nouveaux agents actifs ayant un effet sur le rythme jour/nuit et sur le sommeil - Google Patents

Procede d'utilisation de constructions transfectables composees de luciferase et du promoteur du gene periode 1 humain pour l'identification de nouveaux agents actifs ayant un effet sur le rythme jour/nuit et sur le sommeil Download PDF

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Publication number
WO2001079539A2
WO2001079539A2 PCT/EP2001/004383 EP0104383W WO0179539A2 WO 2001079539 A2 WO2001079539 A2 WO 2001079539A2 EP 0104383 W EP0104383 W EP 0104383W WO 0179539 A2 WO0179539 A2 WO 0179539A2
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WO
WIPO (PCT)
Prior art keywords
vip
pacap
sleep
substances
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2001/004383
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German (de)
English (en)
Other versions
WO2001079539A9 (fr
WO2001079539A3 (fr
Inventor
Dirk Motzkus
Erik Maronde
Sabine Loumi
Urs Albrecht
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
IPF Pharmaceuticals GmbH
Original Assignee
IPF Pharmaceuticals GmbH
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Filing date
Publication date
Application filed by IPF Pharmaceuticals GmbH filed Critical IPF Pharmaceuticals GmbH
Priority to AU2001268973A priority Critical patent/AU2001268973A1/en
Publication of WO2001079539A2 publication Critical patent/WO2001079539A2/fr
Publication of WO2001079539A3 publication Critical patent/WO2001079539A3/fr
Anticipated expiration legal-status Critical
Publication of WO2001079539A9 publication Critical patent/WO2001079539A9/fr
Ceased legal-status Critical Current

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Classifications

    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/204—IL-6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2278—Vasoactive intestinal peptide [VIP]; Related peptides (e.g. Exendin)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2864—Sleep disorders

Definitions

  • the present invention relates to a construct of a promoter of the periodl gene and luciferase, a method for the identification of substances which influence the periodl gene and the use of these substances.
  • the circadian clock is a self-sustaining oscillator with a periodicity of approx. 24 hours.
  • the oscillator is controlled by a number of genes, the central components of which are called period (PER) genes.
  • the human periodl (hPERl) in particular seems to play an important role in adapting to changing environmental conditions.
  • the object is achieved by a transfectable construct from the promoter of the human periodl gene and luciferase.
  • This construct is referred to below as hPERl-luc.
  • the construct is transiently or stably transfected into a human cell line. These cells can then be brought into contact with test substances in the presence of luciferin. The then measurable chemiluminescence is proportional to the expression of the luciferase and thus shows the effect of the tested substances on the promoter of the hPERl.
  • the hPERl-luc construct can be transiently or stably transfected in human cell lines (eg HUH-7, SNB-19 or SH-SY5Y).
  • the transfected cells are seeded on 96-well plates with white walls and left overnight. The next day, the cells are um, which contains 1 mM sodium luciferin with the appropriate control substances or peptide fractions.
  • the plate is measured every 1 to 4 hours in a chemiluminescence reader for up to three days. Between the measurements, the plate is placed in an incubator.
  • the hPERl-luc construct according to the invention is particularly suitable for the identification of sleep-modulating and neuroactive substances.
  • the construct can also be used for screening, i.e. can be used to test a large number (ten or more) of substances or mixtures of substances.
  • Human peptide banks are particularly suitable for the screening, i.e. Substance libraries containing a large number of peptides in partially or completely purified fractions. Corresponding peptide banks can be, for example, hemofiltrate banks or placenta banks.
  • the subject of the invention is. also a method for screening substance libraries, in which cells which are transfected with the construct hPERl-luc according to the invention are brought into contact with substances from the substance library, and the expression of the luciferase is tested.
  • the hPERl-luc construct according to the invention can also be used in a method for identifying substances which delay the circadian rhythm of humans (phase retarders) or accelerate (phase accelerators).
  • the invention also relates to a method for identifying sleep-modulating and neuroactive substances, cells being transfected with a construct from the promoter of the human periodl gene and a reporter gene, incubated with substances and expression of the reporter gene being determined.
  • luciferase such as "firefly” or "renilla” luciferase
  • other suitable reporter genes are intra- cellular or excreted alkaline phosphatase, green fluorescent protein or derivatives of the protein (blue, yellow, red fluorescent protein), beta-galactosidase (beta-Gal; lacZ) or chloramphenicol acetyltransferase (CAT).
  • VIP vasoactive intestinal polypeptide
  • PACAP pituitary-derived adeylate cyclase activating peptide
  • These substances can be used as phase accelerators. They are therefore suitable for the therapy of sleep-associated diseases.
  • Such sleep-associated diseases include, for example, the so-called “jet lag”, which can occur due to a change in time after long-haul flights, consequences of persistent shift work and so-called “seasonal affect disorders” (SAD) [Buney, W.E. and Bunney, B.G. (2000) Neuropsychopharmacology 22, 335-345].
  • SAD seasonal affect disorders
  • Such diseases include symptoms that can also be treated individually with the substances according to the invention, in particular daytime tiredness with hypersomnia, sleep disorders, obesity, carbohydrate craving, symptoms similar to wintering, depression, psychoses, changes in melatonin - and cortisone secretion and shortening of the REM sleep phase.
  • Suitable derivatives of VIP or PACAP which can also be used according to the invention, are particularly suitable
  • PACAP 1-38 hsdgiftdsysryrkqmavkkylaavlgkrykqrvknk PACAP 1-27 hsdgiftdsysryrkqmavkkylaavl PACAP 1-26 hsdgiftdsysryrkqmavkkylaav PACAP 1-25 hsdgiftdsysryrkqmavkkylaa PACAP 1-24 hsdgiftdsysryrkqmavkkyla PACAP 1-23 hsdgif dsysryrkqmavkky1 VIP 1-28 hsdavftdnytrlrkqmavkkylnsiln VIP 1-27 hsdavftdnytrlrkqmavkkylnsil VIP 1-26 hsdavftdnytrlrkqmavkkylnsi VIP 1-25 hsd
  • oncostatin M XP_009916 leukemia inhibitory factor (LIF): XP_009915 cardiotrophin-1 (CT-1): Q16619 ciliary neurotrophic factor (CNTF): P26441
  • IL-1 beta human interleukin 1 beta
  • Figure 1 shows the putatively responsive elements of the hPERl promoter.
  • the scaling relates to the position of the first base of the putative element in hPERl-Hindlll (NCBI No. AF284444); the specified positions in parentheses at the putative transcription start point.
  • Figure 2 shows a schematic way of producing the hPERl-luc construct.
  • Figure 3 shows the induction of luciferase by forskolin.
  • FIG. 4 shows the induction of the hPERl-luc construct by adding a cAMP analog.
  • FIG. 5 shows the endogenous increase in the transcription of hPERl by adding the cAMP analogue and PMA.
  • FIG. 6 shows the stimulation of hPERl-luc transfected JEG-3 cells with VIP and PACAP.
  • Figure 7 shows the induction of hPERl-luc by IL-6.
  • the hPERl-luc is cloned starting from the plasmid hPERl-SuperCos I (39C2, Sun et al., Cell 90 (1997) 1003-1011).
  • the cosmid was cut with various restriction enzymes and the resulting bands hybridized with a PER1 probe.
  • the hPERI-HindIII fragment identified in this way was cloned into pBluescript (pBRS). Then there was a Subcloning into pEGFP-NI (pEGFP), from whose vector sequence the Nhel interface was used.
  • This vector as well as the promoterless pGL2-basic vector, were modified so that one end of the fragment had a free Nhel site (sticky) and the other side showed no overhanging ends (blunt).
  • One way of construction is shown in FIG. 2.
  • Human hepatoma cells (HUH-7) were in Dulbecco's minimum essential medium (DMEM) with 10% fetal bovine serum in the presence of penicillin / streptomycin (100 ⁇ / ml) and 2 mM L-glutamine at 37 ° C at a 5 % CO 2 atmosphere cultivated.
  • DMEM Dulbecco's minimum essential medium
  • penicillin / streptomycin 100 ⁇ / ml
  • 2 mM L-glutamine at 37 ° C at a 5 % CO 2 atmosphere cultivated.
  • the transfection was carried out according to the regulations of the manufacturer Qiagen.
  • the test is carried out in each case of 40,000 cells per well, in which the cells were incubated for 16 hours and then stimulated for 4 hours in a total volume of 25 ⁇ i.
  • the substances to be examined were preincubated for 20 min and then 25 ⁇ l steady glo solution (from Promega) were added to each well and the cells were lysed. The light emission was measured with a luminometer.
  • FIG. 4 shows the influence of different concentrations of the cAMP analog in the presence of PMA (phorbol 12-myristate 13-acetate).
  • FIG. 5 shows the increase in expression determined by this method by incubation with 100 mM Sp-5,6-DCI-cBiMPS and 1 mM PMA for two hours.
  • the hPERl-luc construct according to the invention was transiently transfected into human choriocarcinoma cells (JEG-3). After 24 hours, the cells were plated in 24-well dishes and incubated with the substances VIP, PACAP (1-27) and PACAP (1-38). The peptides show a 4- to 5-fold stimulation of the reporter gene in the luciferase assay (FIG. 6).
  • Example 7
  • Interleukin-6 was investigated as a further substance alone or together with constant amounts of Sp-5,6-DCI-cBiMPS. The results are shown in Figure 7.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Genetics & Genomics (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
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  • Immunology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Urology & Nephrology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Plant Pathology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Vascular Medicine (AREA)
  • Endocrinology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une construction transfectable composée du promoteur du gène de période 1 humain et de luciférase (hPER1-luc).
PCT/EP2001/004383 2000-04-18 2001-04-18 Procede d'utilisation de constructions transfectables composees de luciferase et du promoteur du gene periode 1 humain pour l'identification de nouveaux agents actifs ayant un effet sur le rythme jour/nuit et sur le sommeil Ceased WO2001079539A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001268973A AU2001268973A1 (en) 2000-04-18 2001-04-18 Method for using transfectable constructions from luciferase and from the promotor of the human period 1 gene for identifying novel active substances that influence the day/night rhythm and sleep

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10019360.9 2000-04-18
DE10019360 2000-04-18

Publications (3)

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WO2001079539A2 true WO2001079539A2 (fr) 2001-10-25
WO2001079539A3 WO2001079539A3 (fr) 2002-05-10
WO2001079539A9 WO2001079539A9 (fr) 2003-05-22

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PCT/EP2001/004383 Ceased WO2001079539A2 (fr) 2000-04-18 2001-04-18 Procede d'utilisation de constructions transfectables composees de luciferase et du promoteur du gene periode 1 humain pour l'identification de nouveaux agents actifs ayant un effet sur le rythme jour/nuit et sur le sommeil

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1248107A3 (fr) * 2001-03-22 2002-10-16 IPF Pharmaceuticals GmbH Procédé et appareil de criblage à résolution temporelle
CN115487299A (zh) * 2021-12-31 2022-12-20 中国人民解放军军事科学院军事医学研究院 初级纤毛调控生物节律及其相关应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2260763A1 (fr) * 1996-07-18 1998-01-29 The General Hospital Corporation Regions regulatrices du gene du recepteur 1a de la melatonine et utilisations correspondantes
WO1999012952A1 (fr) * 1997-09-09 1999-03-18 Research Development Foundation Gene de mammifere de type rythme circadien

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1248107A3 (fr) * 2001-03-22 2002-10-16 IPF Pharmaceuticals GmbH Procédé et appareil de criblage à résolution temporelle
CN115487299A (zh) * 2021-12-31 2022-12-20 中国人民解放军军事科学院军事医学研究院 初级纤毛调控生物节律及其相关应用
CN115487299B (zh) * 2021-12-31 2023-06-13 中国人民解放军军事科学院军事医学研究院 初级纤毛调控生物节律及其相关应用

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AU2001268973A1 (en) 2001-10-30
WO2001079539A3 (fr) 2002-05-10

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