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WO2000026362A2 - Proteines de la famille de la stomatine et leur utilisation comme proteines cibles pour le traitement de la douleur - Google Patents

Proteines de la famille de la stomatine et leur utilisation comme proteines cibles pour le traitement de la douleur Download PDF

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Publication number
WO2000026362A2
WO2000026362A2 PCT/DE1999/003434 DE9903434W WO0026362A2 WO 2000026362 A2 WO2000026362 A2 WO 2000026362A2 DE 9903434 W DE9903434 W DE 9903434W WO 0026362 A2 WO0026362 A2 WO 0026362A2
Authority
WO
WIPO (PCT)
Prior art keywords
stomatin
proteins
use according
development
family
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/DE1999/003434
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German (de)
English (en)
Other versions
WO2000026362A3 (fr
Inventor
Anne Gabriele Mannsfeldt
Gary Richard Lewin
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.)
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Original Assignee
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft filed Critical Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Publication of WO2000026362A2 publication Critical patent/WO2000026362A2/fr
Publication of WO2000026362A3 publication Critical patent/WO2000026362A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Proteins of the stomatin family and their use as target proteins for pain therapy are also known.
  • the invention relates to proteins of the stomatin family and their use as target proteins for pain therapy.
  • Red blood cells contain a lot of proteins, both inside the cell and in the cell envelope (cell membrane); one of the main membrane proteins is stomatin. It is absent in patients suffering from overhydrated stomatocytosis. It has long been known that in these patients the red blood cells have an increased permeability of their cell membranes to sodium and potassium. Because of this, it has been postulated that stomatin regulates an - unidentified - ion channel in the cell membrane. Research on stomatin therefore focuses on the function in erythrocytes. The human stomatin cDNA was cloned independently in 1991/92 by three groups (Stewart et al. (1992) Blood 79: 1593-1601).
  • mec-2 is involved in the conversion of a mechanical stimulus into a receptor potential (mechanotransduction). This enables the worm to perceive a touch stimulus (Huang et al, (1995) Nature 378: 292-295).
  • mec-2 connects an ion channel to the cytoskeleton, thus opening the ion channel through a mechanical stimulus.
  • Mec-2 has a high sequence homology to stomatin (66% in the central 260 amino acids).
  • the object of the invention is to identify and isolate further members of the stomatin family and to develop medical applications for this protein family. This object is achieved according to the claims.
  • the invention relates to stomatin and new proteins of the stomatin family, in particular N-stomatin, comprising 287 amino acids and its fragments, mutants and polymorphisms, its coding cDNA with a length of 1847 nucleotides and their fragments, mutants and polymorphisms and the use as target proteins for pain therapy . Furthermore, it relates to the use for the development of pain therapeutics, analgesics / anesthetics, for the construction of genes, for the development of pharmaceutically relevant substances and for diagnostic kits.
  • the invention is based on our own knowledge that stomatin has an important function in mechanotransduction in mammals. Stomatin occurs in the nerve cells that are responsible for the perception of touch and pain stimuli. These cells are called sensory neurons. It was found that the sensitivity to mechanical stimuli in mice that lack stomatin (knockout mice) is severely impaired.
  • an in vitro skin-nerve preparation was used, which makes it possible to irritate the skin with defined stimuli and to measure the response of the nerves in the form of action potentials.
  • a neuron responds to a weak stimulus with few action potentials, stronger stimuli trigger higher frequencies of action potentials. This transmits the strength of the stimulus to the brain.
  • AM receptors A-Mechnanonociceptors
  • Stomatin RO mice the AM receptors (A-Mechnanonociceptors), which are responsible for the perception of pain caused by strong mechanical stimulation of the skin, are clearly disturbed in their function in the Stomatin RO mice. They are unable to respond to stronger touch stimuli with higher frequencies of action potentials. Pain perception is disturbed in mice that lack stomatin.
  • stomatin is an important target for pain therapy: if the function of stomatin is disturbed, the pain sensation is reduced.
  • An essential part of the invention is that a protein analogous to stomatin has been found and isolated.
  • the complete coding sequence was cloned using RACE (Rapid Amplification of cDNA Ends).
  • This cDNA has a length of 1847 base pairs (sequence II).
  • the largest open reading frame contains 864 base pairs and codes for a protein of 287 amino acids in length (sequence I).
  • Stomatin and the analog protein called N-stomatin are 67% identical. In situ hybridizations were used to perform expression analysis.
  • N-stomatin Since the RNA for N-stomatin is formed by all (with a few exceptions) neurons of the nervous system, N-stomatin stands for neuronal stomatin. It was also found that N-stomatin is most strongly expressed in the nervous system but to a lesser extent in various other parts of the body. Because of its high homology to stomatin, N-stomatin also has a function in mechanotransduction and pain sensation and can therefore also be used as a target protein for pain therapy according to the invention.
  • N-stomatin Since stomatin is particularly involved in the warning of strong mechanical stimuli, it is likely that N-stomatin has a function in the perception of weak touch stimuli. With almost all forms of chronic pain or other diseases, even a light touch is felt to be painful. This symptom of touch-induced pain is called allodynia. Since N-stomatin is likely to be involved in the perception of light touches, this protein according to the invention is the target protein for therapeutic agents for the treatment of allodynia.
  • both stomatin and N-stomatin are important for the effect of volatile anesthetics on the body.
  • the mode of action of volatile anesthetics at the molecular level has so far hardly been investigated. It is only known that they act on the neurons of the central nervous system. Such a function can be assumed especially for N-stomatin, since it is expressed by all neurons of the central nervous system (Stomatin, however, only by a few).
  • mice were anesthetized with CO 2 and killed by decapitation.
  • the skin of the right hind leg was extracted together with the saphenous nerve, an afferent nerve that innervates this area of the skin.
  • the skin was in one Organ chamber with hairy side down. This chamber was permanently filled with 32 ° C warm SIF buffer (synthetic interstitial fluid; 2mM CaCl 2 , 5.5mM glucose, 10mM Hepes, 3.5mM KC1, 0.7mM MgS0 4 , 123mM NaCl, 1.5mM NaH 2 P0 4 , 9.5mM Na Gluconate, 7.5mM sucrose).
  • SIF buffer synthetic interstitial fluid
  • the nerve was guided into a lead chamber, which is connected to the organ chamber, and split into finer bundles of axons using fine forceps. For extracellular derivation, these were placed in succession on a silver electrode. If you stimulate the receptive field of one of the neurons whose axon is currently touching the lead electrode, you can determine the properties of a single sensory neuron. First, the receptive field was stimulated electrically and the delay until an action potential was triggered was measured. The line speed was calculated from this time delay and the distance between the receptive field and the lead electrode. The receptive field was then stimulated with von Frey hair in order to determine the activation threshold. Finally, the stimulus response function was measured. For this purpose, the receptive field of the neuron was stimulated with permanent stimuli of various strengths by placing the rounded end of a fine metal rod on the receptive field for 10 seconds with the help of a micrometer screw.
  • the specific primers tcacgcagggaaacacaat and tcagctggaggcgatgag were used for them.
  • the first PCR was carried out with the Oligo dT Anchor Primer and the specific primer agctgagacggcgctgtagattc.
  • the PCR anchor primer and the specific primer gtagacgactccatccacttg were used for the second ('nested') PCR.
  • the 550 bp fragment resulting from this second PCR was cloned and sequenced and assembled with the EST clone. Based on this sequence, a second RACE experiment was carried out.
  • the specific primer CTC GGG TGA ATC CAT CTC ATT was used in the first PCR.
  • the sequence for the second specific primer was: CAG ACG TTT GTC AAA TCT CGA.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne la stomatine et de nouvelles protéines de la famille de la stomatine, notamment la N-stomatine contenant 287 acides aminés, son ADNc codant ayant une longueur de 1847 nucléotides. L'invention concerne également leur utilisation comme protéines cibles pour le traitement des douleurs et leur utilisation pour la production d'agents thérapeutiques pour le traitement de la douleur, analgésiques/anesthésiques, pour la constitution de gènes, pour le développement de substances d'importance pharmaceutique et dans kits de diagnostic.
PCT/DE1999/003434 1998-10-30 1999-10-28 Proteines de la famille de la stomatine et leur utilisation comme proteines cibles pour le traitement de la douleur Ceased WO2000026362A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE1998150015 DE19850015A1 (de) 1998-10-30 1998-10-30 Proteine der Stomatinfamilie und ihre Verwendung als Zielproteine für die Schm erztherapie
DE19850015.7 1998-10-30

Publications (2)

Publication Number Publication Date
WO2000026362A2 true WO2000026362A2 (fr) 2000-05-11
WO2000026362A3 WO2000026362A3 (fr) 2000-06-22

Family

ID=7886124

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE1999/003434 Ceased WO2000026362A2 (fr) 1998-10-30 1999-10-28 Proteines de la famille de la stomatine et leur utilisation comme proteines cibles pour le traitement de la douleur

Country Status (2)

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DE (1) DE19850015A1 (fr)
WO (1) WO2000026362A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004028555A1 (fr) * 2002-09-29 2004-04-08 Yeda Research And Development Co. Ltd Proteines se fixant a la resistine, leur preparation, et leurs utilisations
WO2004040299A3 (fr) * 2002-10-30 2004-07-15 Max Delbrueck Centrum Technique d'identification de composes inhibiteurs de la transduction mecanique dans les neurones
US7820876B2 (en) 2000-09-04 2010-10-26 Institut Pasteur Mouse mutant for expression of the alpha6 subunit of the nicotinic acetylcholine receptor

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5763589A (en) * 1997-01-09 1998-06-09 Incyte Pharmaceuticals, Inc. Human membrane protein
CA2302644A1 (fr) * 1997-11-13 1999-05-27 Genset S.A. Adnc etendus pour proteines secretees

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7820876B2 (en) 2000-09-04 2010-10-26 Institut Pasteur Mouse mutant for expression of the alpha6 subunit of the nicotinic acetylcholine receptor
WO2004028555A1 (fr) * 2002-09-29 2004-04-08 Yeda Research And Development Co. Ltd Proteines se fixant a la resistine, leur preparation, et leurs utilisations
WO2004040299A3 (fr) * 2002-10-30 2004-07-15 Max Delbrueck Centrum Technique d'identification de composes inhibiteurs de la transduction mecanique dans les neurones

Also Published As

Publication number Publication date
WO2000026362A3 (fr) 2000-06-22
DE19850015A1 (de) 2000-05-04

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