WO2004081042A2 - Identification de genes impliques dans la douleur de la famille des snsr (recepteur specifique des neurones sensoriels) - Google Patents

Identification de genes impliques dans la douleur de la famille des snsr (recepteur specifique des neurones sensoriels) Download PDF

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WO2004081042A2
WO2004081042A2 PCT/EP2004/002384 EP2004002384W WO2004081042A2 WO 2004081042 A2 WO2004081042 A2 WO 2004081042A2 EP 2004002384 W EP2004002384 W EP 2004002384W WO 2004081042 A2 WO2004081042 A2 WO 2004081042A2
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snsr
nucleic acid
cell
sequences
amino acid
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PCT/EP2004/002384
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German (de)
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WO2004081042A3 (fr
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Clemens Gillen
Kay Hofmann
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Grünenthal GmbH
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    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • 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)

Definitions

  • the present invention relates to genes from receptors of the SNSR ("Sensory Neuron Specific Receptor") family which are associated with pain conditions, in particular chronic pain.
  • the present invention relates to methods for identifying compounds which modulate the function of the new SNSR receptors in order to thus find pain-relevant substances.
  • the invention further relates to the use of such compounds as well as antibodies and antisense nucleic acids against the new receptors of the SNSR family in medicines and diagnostics for the treatment or diagnosis of pain or related disease states.
  • Pain research in recent years has provided the basic knowledge that the development of chronic pain conditions in particular changes in the nervous system, particularly in the nociceptive neurons of the dorsal root ganglia (DRG) and the neurons in the area of the dorsal horns of the spinal cord (see Coderre et al., 1993; Zimmermann & Herdegen, 1 96).
  • Neuronal plasticity is accompanied by changes in the expression of certain genes and leads to long-lasting changes in the phenotype of the affected neurons.
  • the concept of neuronal plasticity has so far been applied primarily to development, learning and regeneration processes, but recent findings from pain research show that this concept also applies to pathophysiological processes takes effect (Tolle, 1997).
  • the identification of genes that are selectively expressed in pain-relevant neurons is a promising strategy to provide new selective analgesics.
  • the neurons present in the spinal ganglia and here in particular the C-fiber neurons and the A ⁇ -fiber neurons are the subject of intensive research, since a number of indications speak for such a procedure.
  • the nociceptors are the first link in the chain of pain perception. Therefore pain can be combated by modulating such nociceptors at the source.
  • the nociceptors play a crucial role, especially in chronic pain, since the chronification is largely due to dynamic changes in the spinal ganglion neurons (eg peripheral sensitization).
  • the nociceptors Due to the specific task as detectors of nociceptive stimuli, the nociceptors express a unique repertoire of ionic channels and receptors that only occur in these neurons and can therefore be expected to have low-side-effects therapy during pharmacological intervention.
  • genes that are exclusively or predominantly expressed in the DRG neurons and that are the subject of intensive research as interesting molecular targets for analgesics (e.g. the capsaicin receptor TrpVI, the purinergic receptor P2X3, the tetrodotoxin-resistant sodium channel Nav1.8, the kainate receptor GluR5, the NGF receptor TrkA, the neurotrophin GDNF, the neuropeptide CGRP etc.).
  • the spinal ganglia as a point of attack for potential analgesics have the advantage that they lie outside the blood-brain barrier and are therefore more easily accessible for medication and also allow separation of possible CNS side effects.
  • a number of clinically relevant pain relievers largely exert their effect by inhibiting primary afferents, as the following table shows:
  • SNSR sensor neuron specific receptors
  • MRGX Mas-related genes type X
  • Gene probes were used in both mice (Dong et al., 2001) as well as in rats and humans (Lembo et al., 2002) examined the tissue expression and found an exclusive expression in all three species in dorsal spinal ganglia and trigeminal ganglia. In-depth analyzes showed that the SNSR are only expressed in the IB4-positive small no ⁇ iceptive neurons (Dong et al., 2001; Lembo et al., 2002).
  • BAM22 Bovine Adrenomedullary peptide 22
  • hSNSR4 16 nM
  • hSNSRS 13 nM
  • BAM22 is a breakdown product of the pain-relevant pro-protein proenkephaiin and can therefore be seen as a further indication that the receptors of the SNSR family play a role in the endogenous modulation of pain sensation.
  • the receptors of the SNSR family are G-protein coupled receptors. Almost half of all drugs on the market are ligands for G-protein-coupled receptors (GPCR).
  • the size of the SNSR gene family is surprisingly divergent in the human, mouse and rat species analyzed so far Six human members of the family are described in Lembo et al. (2002) and designated as SNSR1-6. In Dong et al. (2001) four human proteins of the SNSR family are described and are referred to there as MRGX1-4 The six SNSR sequences and four MRGX sequences described in the publications mentioned are not completely identical, although in some cases very similar. In Dong et al. (2001), 17 murine MRGX genes are also described, along with a further 33 pseudogenes. Lembo et al. ( 2002) describe only one SNSR in the rat and despite repeated attempts, no other receptors were identified in the rat.
  • the present invention is therefore based on the object of providing further members of the SNSR gene family, in particular in order to make them accessible as targets for pain therapeutic agents. This object is achieved by the embodiments of the present invention characterized in the claims.
  • a method for identifying compounds, in particular pain-relevant compounds which modulate the extra and / or intracellular function of SNSR receptors, in particular the rat SNSR-L2 and / or the SNSR-L3 receptor, comprising the Steps:
  • a screening method which, on the basis of the identification of compounds which influence the extra- and / or intracellular function of SNSR receptors, for example SNSR-L2 and / or SNSR-L3, is used to identify substances that have a potential pain effect.
  • the screening method of the present invention is therefore based on the fact that a potential pain effectiveness of a substance or compound via its interaction with the peptide or protein structures relevant according to the invention, which are a section of or a complete SNSR receptor, in particular SNSR -L2- and / or SNSR-L3 receptor (s), can be found.
  • a chemical compound according to the invention is, for example, an organic chemical compound, in particular a low-molecular species, for example with a molecular weight of ⁇ 5000, in particular ⁇ 3000 , especially ⁇ 1500 and is typically physiologically well tolerated. If necessary, it will be part of a composition with at least one further active ingredient and preferably auxiliaries and / or additives and can be used as a medicament.
  • the organic molecule will be particularly preferred if the binding constant for binding to a protein according to the invention is at least 10 7 mol "1 .
  • the compound according to the invention will preferably be such that it can cross the cell membrane, be it by diffusion or via (intra) membrane transport proteins.
  • Other substances according to the invention are biological-chemical compounds such as nucleic acids, in particular DNA or RNA and their respective building blocks, fats, and their constituents, sugars, be they mono-, oligo- or polysaccharides, peptides, in particular oilgo- or polypeptides, or proteins such as enzymes , Antibodies, etc.
  • a "substance" within the meaning of the present invention can of course be composed of several identical or different of the aforementioned species or be a mixture thereof.
  • test substance's ID 50 value preferably an analgesic effect
  • the modulation of the extra- or intracellular function of the SNSR receptors according to the invention can be an enhancement as well as a reduction until the abovementioned functions are completely switched off.
  • complete or partial agonists, antagonists, inverse agonists and allosteric modulators of the SNSR receptors can be identified with the method according to the invention.
  • the "cell system” in which a polypeptide according to the invention or a section thereof is expressed can, on the one hand, be an intact cell, for example from possibly immortalized cell lines, primary cell lines or other cell lines, or the cell can originate from a tissue, the cell assembly being used for Isolation of the cells is mostly dissolved.
  • the cell system in the method according to the invention can be a system that originated from an originally intact cell, this usually being a corresponding preparation from cells.
  • the "preparation” includes, in particular, homogenates of cells, e.g. corresponding cell lysates, e.g. the cytosol, a membrane fraction of the cells with membrane fragments, a suspension of isolated cell organelles etc.
  • step (b) of the identification method according to the invention the cell system is brought into contact with a test substance, which generally includes a corresponding incubation of the cell system in the presence of the test compound.
  • the contacting or incubation is typically carried out in an aqueous medium for a defined time, so that the test substance can or may react with the polypeptide (s) or peptide (s) expressed by the cell system in step (a) in any way influences, ie modulates, the extra- or intracellular function of the expressed peptides or polypeptides.
  • the preferably aqueous medium can be tempered, for example between 4 ° C.
  • the duration of the contacting ie the incubation time, can be varied between a few seconds and several hours, but also up to several days or even weeks, the type and manner of interaction of the substance with the cell system, in particular the one therein expressed peptide (s) or protein (s) according to the invention will have to be taken into account.
  • Preferred incubation times are, for example, between 1 min and 60 min.
  • the medium in particular the aqueous medium, can contain suitable salts and / or buffer systems, so that, for example, a pH of between 6 and 8, preferably pH 7.0 to 7.5, prevails in the medium during the incubation.
  • Suitable buffer systems are, for example, acetate, phosphate and Tris-HCl buffers, to which salts, in particular NaCl, KCl, CaC, MgCl 2 , etc., are usually added in order to set a suitable ionic strength.
  • Buffer systems used with preference are, for example, phosphate-buffered saline (PBS) or tris-buffered saline (TBS). Suitable substances such as coenzymes, nutrients, growth factors etc. can also be added to the medium.
  • the suitable conditions can be determined by a person skilled in the art depending on the interaction of the substance to be examined with the peptide (s) or protein (s) or on the type of modulation of the peptide or protein or peptides or proteins of the literature and / or fewer, simple preliminary tests can be easily determined in order to obtain a measurement value that is as clear as possible in step (c) in the method according to the invention.
  • the proteins or sections thereof expressed in the cell system in step (a) of the method according to the invention play on account of their belonging to the SNSR family, the majority of which express their members in dorsal spinal ganglia and trigeminal ganglia, in particular the IB4-positive small nociceptive neurons be in the endogenous modulation of the Pain sensation matters. Therefore, they are particularly interesting as a point of attack for potentially analgesic substances.
  • the method of the present invention can be used to find pain-relevant substances which are used in the methods previously known in the prior art, cannot be identified, in particular with other peptides or proteins, which represents a considerable advantage of the method according to the invention.
  • sections of nucleotide sequences coding for corresponding polypeptides or proteins can be used, but this should be at least one section coding for at least 10, preferably 20, more preferably at least 40, most preferably at least 60 amino acids of the polypeptide or protein , Accordingly, only partial sections of at least 10, preferably 20, more preferably at least 40, most preferably at least 60 amino acids of one of the polypeptides according to the invention can be used for a screening method.
  • Peptides and proteins which are encoded by a nucleotide sequence which represents a fragment, a derivative or an allele or a mutant of the sequences coding for the abovementioned polypeptides or of the sequences shown in the abovementioned figures can also be used.
  • a mutant or an allele or a derivative of a sequence according to the invention results in particular from the deletion, addition, insertion and / or substitution of one or more nucleotides of the respective wild-type sequence.
  • Such fragments, derivatives or alleles are preferably one of the depicted Sequences is at least 60%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% homologous. It is essential here that the sequence change is such that the interaction with the peptide (s) or proteins expressed in the cell system and thus the function of the method is not influenced.
  • x% homology means an x% agreement of the base sequence (sequence identity) in the coding region of the polynucleotide.
  • the proteins or (poly) peptides can also be selected from those functionally homologous species which are encoded by a nucleic acid and which are stable and remain stable under standard conditions, preferably under stringent conditions (ie conditions under which only perfectly base-paired nucleic acid strands are formed ), to a nucleic acid with a nucleotide sequence according to positions 179475 to 180485 (or 180488 including stop codon) of FIG. 2, according to positions 132249 to 133316 (or 133319 including stop codon) of FIG. 2, according to positions 33328 to 32417 (or 32414 including stop codon) of FIG. 2, according to FIG. 3 or according to FIG. 5 or their antisense polynucleotide or antisense nucleic acid.
  • homologous or sequence-related nucleotide sequences from all mammalian species, including humans are isolated by conventional methods by homology screening by hybridization with a sample of the nucleic acid sequences according to the invention or parts thereof.
  • Functional equivalents are also to be understood as homologs of the native sequences, for example the sequences shortened in the above sequences, for example their sequences from other mammals, single-stranded DNA or RNA of the coding and non-coding DNA sequence.
  • short oligonucleotides of the conserved areas which can be determined in a manner known to the person skilled in the art, can be used.
  • the use and function of at least 10, preferably at least 20 amino acid long nucleotide sections (also disclosed as such) of the nucleotide sequences according to the invention are disclosed as primers for PCR reactions or as oligonucleotides on DNA chips, in particular in the form of microarrays.
  • longer fragments of the nucleic acids according to the invention or the complete sequences can also be used for the hybridization.
  • These standard conditions vary depending on the nucleic acid sequence used (oligonucleotide, longer fragment or complete sequence) or on the type of nucleic acid (DNA or RNA) used for the hybridization.
  • the melting temperatures for DNA: DNA hybrids are approx. 10 ° C lower than those of DNA: RNA hybrids of the same length.
  • DNA hybrids are advantageously 0.1 ⁇ SSC and temperatures between approximately 20 ° C. to 45 ° C., preferably between approximately 30 ° C. to 45 ° C.
  • DNA: RNA hybrids the hybridization conditions are advantageously 0.1 ⁇ SSC and temperatures between approximately 30 ° C.
  • These specified temperatures for the hybridization are, for example, calculated melting temperature values for a nucleic acid with a length of approximately 100 nucleotides and a G + C content of 50% in the absence of formamide.
  • the experimental conditions for DNA hybridization are in relevant textbooks of genetics, such as in Sambrook et al. ("Molecular Cloning", Cold Spring Harbor Laboratory, 1989), and can be calculated according to formulas known to the person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrid or the G + C content. The person skilled in the art can obtain further information on hybridization from the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Harnes and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical
  • an “antisense polynucleotide” or an “antisense nucleic acid” according to the present invention is a molecule consisting of several natural or modified nucleic acid building blocks, the base sequence of which is at least partially or partially complementary to the base sequence of a partial area of a naturally occurring species , for example the naturally occurring mRNA. Due to the complementarity, the antisense polynucleotide according to the invention or the antisense nucleic acid according to the invention is capable of performing the target molecule under standard hybridizations, as defined above, preferably under stringent conditions as defined above.
  • a partial protein of at least 10 amino acids long of one of the aforementioned proteins and / or peptides is used, since already 10 amino acids, preferably 15, in particular 20 amino acids, are or can be completely specific.
  • the cell system or the cell from which the cell system, in particular a corresponding cell preparation, of the identification method according to the invention emerges for expressing the section (s) of the polypeptide (s) comprising at least 10 amino acids and / or genetically manipulated to measure the at least one parameter.
  • Genetic material is introduced into the cell or the cell system, in particular one or more polynucleotide sequences.
  • the cell or the cell system is preferably transfected with one or more vector (s), preferably expression vector (s), which are used for the above-defined polypeptides or sections thereof and / or (at least) a G protein and / or (at least) one reporter gene encoded.
  • the genetic engineering manipulation allows the measurement of at least one of the functional parameters changed by the test substances.
  • genetic engineering manipulation creates conditions under which the change in a functional parameter can be measured at all or improved.
  • the genetic engineering manipulation expresses a form of a G protein that is not endogenously expressed in the cell or that a reporter gene is introduced. This means in particular the introduction of an endogenously non-existent or physiologically unexpressed G protein (GTP-binding protein) into the cell, for example the introduction of a chimeric G protein that allows a change in the signaling pathway or a promiscuous G protein who is very loyal.
  • GTP-binding protein an endogenously non-existent or physiologically unexpressed G protein
  • the introduction of a reporter gene in turn allows the measurement of an (extracellularly triggered) induced expression of the gene product.
  • nucleic acid polynucleotide
  • a nucleic acid (polynucleotide) coding for one of the above-defined polypeptides or a section thereof it can be achieved, for example, that a peptide or protein that is not endogenously expressed in the cell or preparation used in the method is is synthesized by the cell. It is particularly preferred if the polynucleotide is contained in a recombinant DNA construct.
  • a (recombinant) DNA construct is understood to mean a DNA molecule produced in vitro, in particular a corresponding vector, preferably an expression vector.
  • step (a) the cell is genetically manipulated, it is preferred that the cell is cultivated after the genetically manipulated state under conditions which allow expression, optionally under selection pressure.
  • “Culturing” means to keep cells or tissue under conditions that ensure the survival of the cells or their successor generation.
  • the conditions here should be chosen so that an expression of the genetic manipulation added material is made possible.
  • the pH, oxygen content and temperature should be kept physiological and sufficient nutrients and necessary cofactors added.
  • the selection pressure makes it possible to continue cultivating only the cells in which the genetic engineering manipulation was at least partially successful. This includes, for example, the introduction of antibiotic resistance via a DNA construct.
  • the cell used is an amphibian cell, bacterial cell, yeast cell, insect cell or an immortalized or native mammalian cell or the cell system is derived from such a cell.
  • amphibian cells are enopt / s oocytes, E. coli cells for bacterial cells, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris etc. for yeast cells, Sf9 cells for insect cells, HeLa cells for immortalized mammalian cells and for native cells Mammalian cells the CHO (Chinese Hamster Ovary) cell.
  • the measurement is carried out by the displacement of a known labeled ligand by the peptide or protein and / or by the activity of a labeled test substance bound to it.
  • a ligand is a molecule which binds to the protein or peptide with a sufficient, in particular high specificity, which is displaced from the binding site when a test substance also binds to this binding site, but possibly with a conformational conversion of the peptide or protein at another site with it interacts so that the known ligand is released. Labeling is to be understood as a chemical modification of the molecule which facilitates or enables detection.
  • Examples are radioactive or luminescent, in particular fluorescent, markings. If the identification method of the present invention is carried out in such a way that a modulation of the SNSR receptors is determined on the basis of an interaction of the test substance with these receptors (or sections thereof), it is preferred if in a further step (c) the binding site of the test substance on the polypeptide (s) or a corresponding section thereof, comprising at least 10 amino acids. This is usually done by a suitable structural biological method, in particular by X-ray crystallographic analysis and / or NMR investigations.
  • the binding site of the substance identified in the test as positive with regard to the interaction can be limited and at according to precise mutation analysis, the amino acids essential for the interaction are determined.
  • mutagenesis techniques which are widespread in the prior art, reference is made, for example, to the disclosure content in this regard in Sambrook et al. 1989 and 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY.
  • the measurement of at least one of the functional parameters changed by the test substance is carried out by measuring the regulation, inhibition and / or activation of receptors, ion channels and / or enzymes, especially via measuring the alteration of gene expression, the lonenmilieus, the pH or the membrane potential, via change in the enzyme activity and / or concentration of the 2 nd messenger.
  • the measurement of the action of the substance via the influence of (other) receptors, ion channels and / or enzymes is recorded, on the other hand, examples of changing parameters such as gene expression, ion environment, pH, membrane potential, which are to be measured as preferred, Enzyme activity or concentration of 2 nd Messenger.
  • changing parameters such as gene expression, ion environment, pH, membrane potential, which are to be measured as preferred, Enzyme activity or concentration of 2 nd Messenger.
  • cAMP cyclic AMP
  • IP3 inositol triphosphate
  • DAG diacylglycerol
  • Knowledge of the primary sequence of the receptors of the SNSR family according to the invention can be used to produce recombinant constructs which take advantage of the properties which have already been characterized and are known from the prior art. For example, certain sequence areas of SNSR proteins according to the invention can be exchanged for certain sequence areas of a known, well-characterized SNSR.
  • the resulting construct can be used to identify with known ligands or agonists, the G-protein coupling, and who used 2 nd messenger systems, or well-known G-protein coupling for the discovery of ligands, particularly agonist ( complete, partial, inverse, etc.) or antagonists and allosteric modulators.
  • chimeric receptors according to the invention for example for the abovementioned uses, can, for example, by a method as described by Kobilka et al. described (and belonging to the disclosure of the present invention), (Kobilka BK, Kobilka TS, Daniel K, Regan JW, Caron MG, Lefkowitz RJ (1988) Chimeric alpha 2-, beta 2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity. Science 240: 1310-1316).
  • constitutively active receptor mutants of the SNSR family can be used according to the invention to characterize the effect of these receptors on signal transduction pathways and for "screening" for ligands.
  • Receptors according to the invention as representatives of the G protein-coupled receptors can be mutated in a certain way to bring about changes the physiological and pharmacological behavior of these receptors. This can be used, for example, to Identify intracellular signaling pathways when the natural ligand or agonist are unknown.
  • a variant of the method according to the invention can be used to identify endogenous or surrogate ligands, which is based on the use of immobilized functional receptors according to the invention.
  • Receptors of the SNSR family according to the invention can be expressed here as fusion proteins with GST, the flag tag or the TAP tag.
  • the corresponding cells are either processed into membranes using standard methods or used directly for solubilization.
  • suitable detergents e.g. Dodecyl maltoside, digitonin, cholate or detergent mixtures
  • the receptors are solubilized and bound to the corresponding affinity matrices such as GST-Sepharose, Anti-Flag-M2-Agarose or IgG-Sepharose etc.
  • the matrices After washing the matrices, they are incubated with tissue extracts or cell supernatants and washed again. If the extract contains an active ligand, e.g. a peptide, it binds to the immobilized receptor and can be identified after elution by analytical methods, e.g. by means of mass spectrometry.
  • an active ligand e.g. a peptide
  • internalization tests represent a further embodiment of the identification method according to the invention in order to be able to identify natural or in particular surrogate ligands for receptors according to the invention, in particular SNSR-L2 or -L3.
  • the different properties of a protein of the GPCR class are also exploited here.
  • the internalization behavior of proteins of the GPCR class can be used. This is to be understood as a regulatory mechanism after activation of the receptor.
  • the advantage of a "screening" method based on this behavior is that a more precise knowledge of the physiology of the respective receptor is not necessary. In particular, there is no need to know about the coupling G proteins and the signal transduction pathways used.
  • Such a test is, for example, by Lenkei et al. (2000, J Histochem Cytochem, 48, 1553-64) and can be used according to the invention analogously for the receptors according to the invention.
  • a C-terminal fusion construct of the protein according to the invention with EGFP is first produced.
  • Stable CHO cells are then produced using standard procedures.
  • Stable clones are selected using a FACS sorter according to EGFP fluorescence. The final selection was made with the help of fluorescence microscopic assessment of the surface expression.
  • the cells are then incubated with HPLC fractions of tissue extracts, and the internalization is determined using a confocal microscope.
  • the evaluation is carried out using morphometric software (NIH Image) based on the principle of the distance of fluorescent signals from the center of the cell. A frequency / distance distribution results in good discrimination for internalization.
  • successive fractionations are advantageously carried out, for example in order to isolate a peptide or protein which interacts appropriately interacting or otherwise (ie indirectly) influencing the receptor of the SNSR family according to the invention (e.g. influencing expression, etc.), and then identified, for example, by sequencing or MALDI-TOF.
  • Another application of the basically same method is described by Ghosh et al. (2000, Biotechniques, 29, 170-5; Conway et al., 1999, J Biomol Screen, 4, 75-86), both of which form part of the present disclosure.
  • a functional Ca test is used for the identification or screening process, which can also be used to characterize receptors according to the invention.
  • use is made of the fact that a large number of 7TM receptors (receptors with 7 transmembrane domains), which are produced in HEK293 cells, in CHO cells or other cells, are coupled to G proteins of the Gq class for activation the PLC and lead to the mobilization of intracellular Ca 2+ .
  • receptors are not to be coupled to G proteins of the Gq class, they can be co-expressed by chimeric G proteins or the G proteins G ⁇ 15 or G ⁇ 16, which are relatively unspecifically coupled to receptors, for signal transmission via PLC, ie to release Ca 2+ .
  • the receptors of the SNSR family according to the invention can be expressed, for example, in HEK293 and in CHO cells both stably and transiently alone and together with the chimeric G protein Gqi5 and alternatively with the G protein G ⁇ 15.
  • the cells are then preferably loaded with a membrane-permeable Ca 2+ -binding fluorescent dye, for example Fura-2 or Fluo-3 or -4 (these and other suitable fluorescent dyes are available, for example, from molecular probes) and, after washing the cells, with different ones Test substances are added and at the same time the Ca 2+ release, ie the intracellular Ca 2+ concentration, is measured, for example using a FLIPR device from Molecular Devices.
  • Test substances which give a positive signal are finally preferably tested in control cells (transfected only with the vector) and, if the signal turns out to be specific, characterized pharmacologically, ie with concentration-response curves.
  • the Ca 2+ response caused by a test substance can also be measured by other Ca 2+ detectors, e.g. via AequoScreen from Euroscreen (Brussels, Belgium; see e.g.http: //www.pharmaceutical- technology.com/contractors/compound_man / € screen /).
  • Cells are used that express the gene of the protein apoaequorin. After loading the cells with coelenterazine, which binds to apoaequorin, aequorin is formed.
  • the Ca 2+ activates the aequorin for the oxidation of coelenterazine, as a result of which light is released.
  • the intensity of the light emission is proportional to the increase in the intracellular Ca 2+ concentration and thus a measure of the activity of the test substance identified as ligand (taking into account the corresponding controls).
  • the receptors are stimulated in the presence of sufficiently high concentrations of various test substances with a known agonist, for example BAM22-6, neuropeptide FF (NPFF) or neuropeptide AF (NPAF).
  • NPFF neuropeptide FF
  • NPAF neuropeptide AF
  • cAMP tests can also be used to characterize the receptors of the SNSR family according to the invention, in particular to identify ligands.
  • the background of this approach according to the invention for the identification of ligands (agonists or antagonists) but also for the pharmacological characterization of receptors of the SNSR family is the property of receptors of the class of the GPCRs, for example of proteins according to the invention, to be able to act either stimulating or inhibiting on adenylate cyclases , usually by activating so-called stimulating Gs or inhibiting Gi proteins.
  • stimulating Gs or inhibiting Gi proteins usually by activating so-called stimulating Gs or inhibiting Gi proteins.
  • the associated change in the cAMP level in the cell can be examined via direct or indirect measurements.
  • the receptor genes or parts thereof are expressed stably or transiently in mammalian cells.
  • GPCRs that activate adenylate cyclases which increases the cAMP level in the cell
  • a potential agonist is identified among the test substances by an increased cAMP concentration compared to control cells.
  • Antagonists among the test substances for example in an HTS approach, are identified by blocking the increase in cAMP concentration caused by an agonist.
  • the adenylate cyclase is stimulated either directly with forskolin or by activation of a Gs-coupled receptor in the test, as a result of which the cAMP level increases.
  • An agonist of the Gi-coupled receptor inhibits this increase.
  • cAMP [3H] test system for direct cAMP measurements, a number of commercially available tests, such as, for example, the cAMP [3H] test system from Amersham, can be used, which are based, for example, on the principle of the competitive displacement of endogenously formed cAMP by the addition of radiolabeling. tes (tritium) cAMP. Indirect cAMP measurements are usually carried out using reporter tests. For this purpose, the receptors are expressed in cell lines which contain reporter systems, for example the CRE luciferase system. cAMP activates the expression of the luciferase, the activity of which is measured by the implementation of appropriate substrates and luminometric measurement of the products. Reporter tests are particularly suitable for mass screening methods.
  • test systems according to the invention for the identification of ligands SNSR proteins or for the characterization of 2 nd messenger systems of these receptors according to the invention possible, the invention in particular for determining the adenylate cyclase activity in cells or membranes - eventually - in addition to the above-described tests according to Salomon (Salomon et al. (1979) Adv. Cyclic Nucleotide Res. 10: 35-55), for determining the inositol-3-phosphate concentration or for measuring a changed release of arachidonic acid.
  • the SNSR proteins can be overexpressed in standard cell lines, and after activation by tissue extracts the activity of the above can be determined nd messenger systems.
  • tests for 2 nd messenger systems of receptors of the GPCR class are known to a person skilled in the art and can be found in the individual case in the literature (see, for example, Signal Transduction: A practical approach, G. Milligan, Ed. Oxford University Press, Oxford, England).
  • Further reporter tests for the screening method according to the invention include MAP kinase / luciferase and NFAT luciferase systems.
  • the activation is of 2 nd messengers invention for identification of ligands, particularly agonists or antagonists that can unfold bind to invention receptors and so their agonistic and / or antagonistic effect of the stimulus formation and forwarding-related pain.
  • Microphysiometers can be used to identify ligands. Signals triggered by ligand binding to a receptor of the SNSR family represent energy-consuming processes. For this reason, such processes are generally carried out with little metabolic changes, including a slight pH shift. These can be detected extracellularly by, for example, a microphysiometer (cytosensor, molecular devices).
  • Ligand binding tests can be carried out according to the invention for their more detailed characterization. Ligand binding tests make it possible to directly measure the pharmacology of a receptor, ie the affinity of various ligands for this receptor.
  • a chemically pure ligand with a high specific activity (30-2000 Ci / mmol) identified or otherwise known by one of the abovementioned methods is typically radioactively labeled, so that the radioactive labeling does not reduce the activity of the ligand with respect to the receptor .
  • the test conditions are optimized both for the use of cells expressing the receptor and of membranes produced therefrom with regard to buffer composition, salt, modulators such as nucleotides or stabilizers such as glycerin, so that the measurement gives a useful signal / background ratio.
  • the specific receptor binding is defined as the difference from the total radioactivity associated with the receptor preparation (cells or membranes), ie measured in the presence of only one specific one, namely the radioligand and the radioactivity, which are present in the presence of both the radioligand and one Excess of non-radioactive labeled ligand is measured.
  • the unlabeled ligand competitively displaces the radioligand. If possible, at least two chemically different competitive ligands are used to determine the non-specific binding. A specific binding that is at least 50% of the total binding is optimal.
  • the binding test is either carried out inhomogeneously as a filtration test or homogeneously as a so-called "scintillation proximity test" or "flash plate test".
  • the preparation (cells or membranes) containing the receptor is incubated with the ligands in a suitable buffer solution until the binding equilibrium has been established, typically 1 h at RT or at 4 ° C. overnight, and then using suitable filters, for example Whatman or Schleicher & Schuell glass fiber filters, which may have been pretreated, for example with polyethyleneimine, are filtered off in order to separate the unbound from the bound radioligand. After washing the filters, they are dried or moistened with a suitable scintillator and the radioactivity contained in the scintillation counter is measured after any necessary incubation.
  • suitable buffer solution typically 1 h at RT or at 4 ° C. overnight
  • suitable filters for example Whatman or Schleicher & Schuell glass fiber filters, which may have been pretreated, for example with polyethyleneimine
  • suitable scintillation beads eg WGA beads
  • WGA beads are incubated with the ligands and receptor-containing membranes in a suitable buffer solution until the binding equilibrium has been established, and then the radioactivity is measured in a suitable scintillation counter. Both binding tests are in HTS format feasible.
  • Solubilized or purified receptors can be measured with the "Scintillation Proximity Test" or with common inhomogeneous tests such as the filtration test after PEG precipitation, the adsorption or the gel filtration test (Hulme E, Birdsall N (1986) Distinctions in acetylcholine receptor activity. Nature 323 : 396-397).
  • a fluorescent ligand e.g. a ligand that has covalently bound a fluorescent dye such as BODIPY can be used.
  • the binding of the fluorescent ligand to the receptor is measured by means of fluorescence polarization.
  • the method is suitable for primary screenings in HTS format as well as for secondary tests.
  • binding sites of the identified and pharmacologically active substances to the SNSR gene products according to the invention are determined using the yeast two-hybrid system or other tests, ie that the amino acids responsible for the interaction, for example also for the interaction between native proteins, are limited, as already mentioned above.
  • high-affinity substances surrogate ligands
  • structural regions responsible for the binding of the native interaction partners
  • the present invention furthermore relates to a nucleic acid comprising a nucleotide sequence, in particular a DNA sequence, which is suitable for a polypeptide with an amino acid sequence according to FIG. 4 or a section thereof comprising at least 10 amino acids and / or for a polypeptide with an amino acid sequence according to FIG 6 or a section thereof comprising at least 10 amino acids, including all functionally homologous derivatives, fragments or alleles.
  • nucleic acid sequences in particular DNA sequences, which contain a sequence region which codes for a polypeptide with an amino acid sequence of a protein of the members SNSR-L2 or -L3 of the SNSR family, in particular the C-terminal (intracellular) Section of a protein according to the invention or a fragment thereof (preferably at least 25 amino acids in length) should be included.
  • DNA sequences are disclosed whose gene product codes for a polypeptide, as shown in one of FIGS. 4 or 6, including all functionally homologous derivatives, alleles or fragments of such a DNA sequence and also infunctional derivatives, alleles, analogues or Fragments (e.g. DN variants) that can inhibit physiological function.
  • DNA sequences hybridizing with these DNA sequences according to the invention are also disclosed.
  • the derivatives, alleles, fragments or analogs of the amino acid sequences according to the invention according to FIGS. 4 and 6 or further native members of the SNSR family preferably retain at least one biological property.
  • the subject matter of the present application also includes partial sequences of the native SNSR sequences according to the invention, in particular the sequences shown in FIG. 3 or 5. These partial sequences typically contain at least 30, more preferably 60, more preferably at least 150 and even more preferably at least 250 nucleotides of fragments of the nucleotide sequences shown in FIGS. 3 and 5. All derivatives, analogs or alleles of the aforementioned partial sequences disclosed are also disclosed. Also the amino acid sequences resulting from these partial sequences according to the invention are also disclosed as such or as a component in larger recombinant proteins. In particular, all conceivable or natively occurring splice variants of the sequences according to the invention are also part of the present disclosure.
  • nucleic acid sequences in particular DNA sequences, which code for a protein which has at least 60%, preferably at least 80%, more preferably at least 90% and even more preferably at least 95% sequence identity (homology) with the sequences according to FIGS 6 has.
  • the nucleotide sequences according to the invention in particular according to FIG. 3 or 5, or their functional or non-functional equivalents, such as e.g. Allele variants or isoforms available.
  • allele variants are understood to mean variants which have 60 to 100% homology at the amino acid level, preferably 70 to 100%, very particularly preferably 90 to 100%.
  • Allelic variants include, in particular, those functional or infunctional variants which can be obtained by deleting, inserting or substituting nucleotides from native SNSR sequences, for example the SNSR-L2 or SNSR-L3 sequences according to the invention, at least one of the essential biological properties preserved.
  • nucleic acid sequences according to the invention in particular also include derivatives of the sequences shown in FIGS. 3 and 5, such as promoter variants.
  • the promoters which are upstream of the specified nucleotide sequences together or individually can by one or more nucleotide exchanges have been changed by insertion (s) and / or deletion (s), the functionality or effectiveness of the promoters being retained or being able to be changed as required.
  • the effectiveness of the promoters can be increased by changing their sequence, or they can be completely replaced by more effective promoters, including organisms of other species.
  • derivatives are also to be understood to mean variants whose nucleotide sequence in the range -1 to -1000 before the start codon have been changed such that the gene expression and / or the protein expression is changed, preferably increased.
  • Derivatives are also to be understood as variants which have preferably been changed at the 3 'end.
  • tags in the literature, for. B. Hexahistidine anchor known or epitopes that can be recognized as antigens of various antibodies (eg also the flag tag) (Studier et al., Meth. Enzymol., 185, 1990: 60-89 and Ausubel et al. (eds.) 1998, Current Protocols in Molecular Biology, John Wiley & Sons, New York), and / or at least one signal sequence for transporting the translated protein, for example into a certain cell organelle or into the extracellular space.
  • nucleic acid construct according to the invention or a nucleic acid according to the invention for example according to FIG. 3 or 5, or their derivatives, variants, homologs or, in particular, fragments can also be expressed in therapeutically or diagnostically suitable form.
  • vector systems or oligonucleotides can be used which extend the nucleic acids or the nucleic acid construct by certain nucleotide sequences and thus code for modified polypeptides which serve, for example, for easier purification, in particular the extension by those described above Tag sequences referenced.
  • nucleic acids which (c) DNA sequences of genomic nucleotide sequences according to the invention, in particular positions 132249 to 133316 (or 133319 including stop codon) of FIG. 2 or positions 33328 to 32417 (or 32414 including stop codon) of FIG. 2, contain or correspond to these.
  • DNA sequences which code for a protein which essentially corresponds to the amino acid sequence according to FIG. 4 or 6 are preferably also disclosed. These DNA sequences contain only a small number of changes compared to the sequences given in the abovementioned figures, for example they can be isoforms. The number of sequence changes will typically not be greater than 10.
  • Such DNA sequences essentially corresponding to the DNA sequences coding for the proteins with the amino acid sequences according to FIGS. 4 and 6, which also code for a biologically active protein can be obtained by generally known mutagenesis methods and the biological activity of the the mutants encoded proteins are identified by screening methods, for example binding studies or the ability to express the biological function, for example in connection with neuronal processes or apoptosis.
  • the corresponding mutagenesis methods include "site-directed" mutagenesis, which provides for the automatically carried out synthesis of a primer with at least one base change.
  • the heteroduplex vector is transferred into a suitable cell system (eg E. coli) and isolated transformed clones isolated.
  • PCR Innis et al. PCR Protocols: A Guide to Methods and Applications
  • Appropriate PCR primers can be used, for example, to introduce new functions into a DNA sequence according to the invention, such as restriction sites, termination codons.
  • sequences according to the invention can be designed accordingly for transfer into cloning vectors.
  • Another object of the present invention relates to a vector, ie a recombinant nucleic acid construct that typically contains a nucleic acid sequence according to the invention as described above, typically a DNA sequence.
  • the vector according to the invention is preferably an expression vector.
  • the nucleic acid sequences according to the invention are functionally linked to at least one genetic regulatory element, such as, for example, transcription and translation signals. Depending on the desired application, this linkage can lead to a native expression rate or to an increase or decrease in the native gene expression.
  • host organisms or host cells can then be transformed, e.g. Cell cultures from mammalian cells.
  • the native regulatory element (s) will typically be used, i.e. the promoter and / or enhancer region of the gene for a protein according to the invention from the SNSR family, in particular for a protein with an amino acid sequence according to FIG. 4 or 6, in particular corresponding regulatory sequences from the rat. Possibly. these native regulatory sequences described above can also be genetically modified in order to produce a changed expression intensity.
  • native regulatory elements according to the invention for other genes can be upstream and / or downstream (5'- or 3'-regulatory sequences) and may also have been genetically modified so that the natural one Regulation is switched off under the control of the abovementioned native regulatory sequences and the expression of the genes — depending on the desire — can thereby be increased or decreased.
  • Advantageous regulatory sequences for the vector according to the invention are for example in Promoters such as cos-, tac-, trp-, tet-, trp-tet-, Ipp-, lac-, Ipp-lac-, laclq-, 17-, 15-, 13-, gal-, trc-, ara- , SP6, l-PR or contained in the I-PL promoter, which are advantageously used in gram-negative bacteria.
  • Promoters such as cos-, tac-, trp-, tet-, trp-tet-, Ipp-, lac-, Ipp-lac-, laclq-, 17-, 15-, 13-, gal-, trc-, ara- , SP6, l-PR or contained in the I-PL promoter, which are advantageously used in gram-negative bacteria.
  • promoters such as amy and SP02
  • yeast promoters such as ADC1, MFa, AC, P-60, CYC1, GAPDH
  • mammalian promoters such as CaM-Kinasell, CMV, Nestin, L7, BDNF, NF , MBP, NSE, beta-globin, GFAP, GAP43, tyrosine hydroxylase, kainate receptor subunit 1, glutamate receptor subunit B included.
  • all natural promoters with their regulatory sequences, such as those mentioned above, can be used for an expression vector according to the invention.
  • synthetic promoters can also be used advantageously.
  • These regulatory sequences are intended to enable the targeted expression of the nucleic acid sequences according to the invention. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
  • the regulatory sequences or factors can preferably have a positive influence on the expression and thereby increase it.
  • the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
  • an increase in translation is also possible, for example, by improving the stability of the mRNA.
  • Regulatory sequences are all elements familiar to the person skilled in the art which can influence the expression of the sequences according to the invention at the transcription and / or translation level.
  • so-called “enhancer” sequences are to be emphasized, which can bring about increased expression via an improved interaction between RNA polymerase and DNA.
  • locus control regions so-called “silencers” or respective partial sequences thereof may be mentioned as further regulatory sequences.
  • These sequences can advantageously be used for tissue-specific expression.
  • So-called terminator sequences are also which are advantageously present in an expression vector according to the invention and, according to the invention, are subsumed under the term “regulatory sequence”.
  • a preferred embodiment of the present invention is the linkage of the nucleic acid according to the invention to a promoter, the promoter typically being located 5 '"upstream" from a DNA sequence according to the invention.
  • Further regulation signals such as, for example, 3'-located terminators, polyadenylation signals or enhancers, can be functionally contained in the expression vector.
  • nucleic acid sequences according to the invention in particular for the sequences according to FIGS. 3 and 5 or for the corresponding proteins, can be contained in one or more copies in a gene construct according to this invention, or, if appropriate, can also be localized on separate gene constructs.
  • expression vector includes both recombinant nucleic acid constructs or gene constructs, as described above, and complete vector constructs, which typically also contain further elements in addition to DNA sequences according to the invention and any regulatory sequences. These vector constructs or vectors are used for expression in a suitable host organism.
  • at least one DNA sequence according to the invention for example a rat gene from the SNSR family, in particular SNSR-L2 or -L3, or for example a partial sequence of such a gene, is inserted into a host-specific vector which provides optimal expression of the genes in the selected host.
  • Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Eds. Pouwels PH et al.
  • vectors are also understood to mean all other vectors known to the person skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus, adenovirus, Sindbis virus, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA. These vectors can auto- nom replicated in the host organism or replicated chromosomally. Linear DNA is typically used for integration into Mammalia.
  • nucleic acid sequences according to the invention can advantageously be increased by increasing the number of gene copies and / or by strengthening regulatory factors which have a positive effect on gene expression.
  • regulatory elements can preferably be amplified at the transcription level by using stronger transcription signals, such as promoters and enhancers.
  • an increase in translation is also possible, for example, by improving the stability of the mRNA or increasing the reading efficiency of this mRNA on the ribosomes.
  • the nucleic acid sequences or homologous genes can be incorporated, for example, into a nucleic acid fragment or into a vector which preferably contains the regulatory gene sequences assigned to the respective genes or promoter activity with an analogous effect.
  • those regulatory sequences are used which increase gene expression.
  • Nucleic acid sequences according to the invention can be cloned together with the sequences coding for interacting or for potentially interacting proteins in a single vector and then expressed in vitro in a host cell or in vivo in a host organism.
  • each of the potentially interacting nucleic acid sequences and the coding sequences according to the invention from the SNSR family can each be brought into a single vector and these can be brought into the respective organism separately using customary methods, such as, for example, transformation, transfection, transduction, electroporation or particle gun become.
  • At least one marker gene (for example antibiotic resistance genes and / or genes which code for a fluorescent protein, in particular GFP) can be contained in an expression vector according to the invention, in particular a complete vector construct.
  • the present invention further relates to host cells which have been transformed with a nucleic acid according to the invention and / or an expression vector according to the invention, in particular a vector construct.
  • all cells are suitable as host cells which allow expression of nucleotide sequences according to the invention (which also includes their alleles or functional equivalents, for example, as derivatives), alone or in combination with other sequences, in particular regulatory sequences.
  • All cells of a pro- or eukaryotic nature can be considered as host cells, for example bacteria, fungi, yeasts, plant or animal cells.
  • Preferred host cells are bacteria such as Escherichia coli, Streptomyces, Bacillus or Pseudomonas, eukaryotic microorganisms such as Aspergillus or Saccharomyces cerevisiae or the common baker's yeast (Stinchcomb et al., Nature, 282: 39, (1997)).
  • Methylotrophic yeasts, in particular Pichia pastoris are advantageously suitable in particular in order to be able to produce relatively large amounts of proteins according to the invention.
  • the receptors are cloned into suitable expression vectors which, for example, also allow expression as a fusion protein with “tag” sequences suitable for purification. After electroporation of the yeasts, stable clones are finally selected. A good description of the method and all the necessary means are provided by the Invitrogen.
  • the expression products can then be functionally characterized and, if necessary, used for "screening" methods according to the invention.
  • cells from multicellular organisms are selected for the expression of DNA sequences according to the invention. This also takes place against the background of a possibly required glycosylation (N- and / or O-coupled) of the encoded proteins.
  • This function can be carried out in a suitable manner in higher rows of eukaryotes compared to prokaryotic cells.
  • any higher eukaryotic cell culture is available as a host cell, although cells from mammals, for example monkeys, rats, hamsters or humans, are very particularly preferred. A large number of established cell lines are known to the person skilled in the art.
  • 293T embryonic kidney cell line
  • BHK hamster kidney cells
  • CHO hamster ovary cells
  • HeLa human cervical carcinoma cells
  • other cell lines in particular those established for laboratory use, such as CHO, HeLa, HEK293, Sf9 or COS cells.
  • Human cells in particular cells of the immune system or adult stem cells, for example stem cells of the blood-forming system (from the bone marrow) are very particularly preferred.
  • the cells or cell lines can express the SNSR receptors according to the invention endogenously as well as exogenously.
  • Human transformed cells according to the invention in particular autologous cells of the patient, are suitable after (above all ex vivo) transformation with nucleotide sequences according to the invention or expression vectors according to the invention, very particularly as a medicament for, for example, gene therapy purposes, that is to say after cell removal, possibly ex vivo Expansion, transformation, selection and final retransplantation.
  • proteins of the SNSR family according to the invention are particularly advantageously produced heterologously in insect cells for functional characterization and for use in “screening” methods according to the invention. Since the concentration of endogenous G proteins in insect cells is relatively low, for example Gi proteins cannot be detected in the "Western blot", and insect cells generally do not express the receptor to be investigated, they are used for in vivo reconstitution of signal transduction pathways of receptors according to the invention.
  • the receptors of the SNSR family are expressed by means of the baculovirus expression system in various insect cell lines, for example Sf9, Sf21, Tn 368 or TN High Five, or MB cells, for example using the BaculoGold Kit from Pharmingen recombinant baculoviruses are produced and the above-mentioned insect cell lines are infected.
  • the cells are infected with the receptor virus and additionally with the viruses expressing the three G protein subunits and appropriate tests, for example cAMP tests, can be carried out the influence of different G protein subunits on the activity of the tors are examined.
  • Insect cells that express receptors or their membranes can also be used in screening tests. Insect cells can easily be multiplied in large amounts in fermenters as well as in shake flasks and are therefore suitable starting material for providing recombinant cell or membrane material for both screening and receptor cleaning.
  • a host cell and an expression vector according to the invention which matches the host cells, such as plasmids, viruses or phages, such as plasmids with the RNA polymerase / promoter system, the phages I, Mu or other temperate phages or transposons, and / or further advantageous ones regulatory sequences form a host cell according to the invention which can serve as an expression system.
  • Preferred expression systems according to the invention based on host cells according to the invention are, for example, the combination of mammalian cells, such as, for example, CHO cells, and vectors, such as, for example, pcDNA3neo vector, or, for example, HEK293 cells and CMV vector, which are particularly suitable for mammalian cells.
  • gene products encoded by the nucleic acid according to the invention.
  • gene products mean both primary transcripts, ie RNA, preferably mRNA, and proteins or polypeptides, in particular in purified form. These proteins regulate or transport signals related to pain in particular.
  • a purified gene product is preferred if it contains a functionally homologous or function-inhibiting (infunctional) allele, fragment, analogue or derivative of this sequence or typically consists of such an amino acid sequence.
  • Functional homology is defined in the sense of the present invention in such a way that at least one of the essential functional properties of the proteins shown in FIGS. 4 and 6 is retained.
  • functionally homologous proteins according to the invention will in particular have a characteristic, for example at least 60%, preferably at least 80%, more preferably at least 90% sequence.
  • a derivative is understood to mean, in particular, those amino acid sequences which have been modified by modifications to their side chains. For example. by conjugation of an antibody, enzyme or receptor to an amino acid sequence according to the invention.
  • derivatives can also be the coupling of a sugar (via an N- or O-glycosidic bond) or fatty (acid) residue (e.g. myristic acid), one or more phosphate groups and / or any modification of a side chain, in particular a free OH group or NH 2 group or at the N or C terminus of an oligo or polypeptide according to the invention.
  • the term “derivative” also includes fusion proteins in which an amino acid sequence according to the invention is coupled to any oligo- or polypeptides.
  • “Analogs” are sequences which are distinguished by at least one amino acid change compared to the native sequence (insertion, substitution). In the context of the present invention, such conservative substitutions are preferred in which the physicochemical character (space filling, basicity, hydrophobicity, etc.) of the exchanged amino acid is retained (polar amino acid, long aliphatic chain, short aliphatic chain, negatively or positively charged amino acid, amino acid with aromatic group).
  • the substitutions can be biologically functional, some. Functional or bio-functional sequences result. For example, arginine residues can be exchanged for lysine residues, valine residues for isoleucine residues or aspartic acid residues for glutamic acid residues.
  • modified in this way compared to the native SNSR proteins typically have at least 60%, preferably at least 70% and particularly adds at least 90% sequence identity to the sequences in the aforementioned figures, calculated according to the algorithm of Altschul et al. (J. Mol. Biol., 215,
  • the isolated protein can advantageously be isolated from the dorsal Sinai ganglia and trigeminal ganglia from Mammalia, in particular Ratu's norwegicus. Homologs from other mammals are also to be understood as functional variants.
  • Analogs are preferred according to the invention if the secondary structure as occurs in the native sequence is also retained in them.
  • less conservative amino acid variations can also be introduced into the native sequence according to the invention. They typically maintain their biological function, in particular as a transducer of a pain-relevant signal. The effect of a substitution or deletion can easily be checked by appropriate investigations, binding tests or, for example, cytotoxic tests.
  • sequences are also included which can produce a so-called dominant-negative effect, that is to say that because of their changed primary sequence they still have binding activity to an extracellular ligand, but cannot pass the signal downstream, ie intracellularly.
  • Analogs of this type therefore function as inhibitors of biological function, in particular as inhibitors of apoptosis.
  • Such analogs are produced by genetic engineering measures, typically by the so-called "site-directed" mutagenesis of a nucleotide sequence which codes for a protein according to the invention (typically sequences according to FIG. 3 or 5).
  • gene products (proteins) according to the invention also include all those gene products (proteins) which are derived from nucleic acid derivatives, fragments or alleles according to the invention of the nucleic acid sequences shown in the figures after transcription and translation.
  • proteins according to the invention can be chemically modified.
  • Glycosyl groups can be added to hydroxyl or amino groups
  • lipids can be covalently linked to the protein according to the invention, as can phosphates or acetyl groups and the like.
  • Any chemical substances, compounds or groups can also be bound to the protein according to the invention by any synthetic route.
  • Additional amino acids e.g. in the form of individual amino acids or in the form of peptides or in the form of protein domains and the like, can with the N- and / or C-terminus of a protein according to the invention.
  • signal or "leader” sequences at the N-terminus of the amino acid sequence of a protein according to the invention are preferred, which lead the peptide cotranslationally or post-translationally into a specific cell organelle or into the extracellular space (or the culture medium).
  • Amino acid sequences can also be present at the N- or at the C-terminus, which allow the binding of the amino acid sequence according to the invention to antibodies as antigen.
  • the flag peptide the sequence of which in the single-letter code of the amino acids is: DYKDDDDK. Or a His tag with at least 3, preferably at least 6, histidine residues. These sequences have highly antigenic properties and thus allow the recombinant protein to be checked and purified quickly.
  • Monoclonal antibodies that bind the flag peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Connecticut.
  • the present invention furthermore relates to at least 10, preferably 20, more preferably at least 30 and even more preferably at least 50 amino acids, partial sections of the native SNSR sequences, in particular the sequences disclosed in FIGS. 4 and 6.
  • Such partial sequences can, for example, be chemically synthesized by methods familiar to the person skilled in the art and can preferably be used as antigens for the production of antibodies.
  • These subsections or their derivatives, alleles or fragments will preferably be disclosed sequences which, in the spatial model of the proteins, form regions which at least partially make up the protein surface.
  • Another object of the present invention is a transgenic non-human organism which has been genetically modified in such a way that it (specifically) changes the amount of at least one amino acid sequence according to the invention, in particular an amino acid sequence according to FIGS. 4 and 4, compared to the normal, ie not changed, organism 6 or FIG. 6, or an amino acid sequence according to the invention which has been specifically modified compared to a native SNSR-L2 and / or SNSR-L3 amino acid sequence, in particular an amino acid sequence according to FIG. 4 or 6, or that the transgenic organism has a native one SNSR-L2 and / or SNSR-L3 amino acid sequence is missing, in particular an amino acid sequence according to FIG. 4 and / or FIG. 6, part of which is missing or changed.
  • an altered amount of at least one gene product according to the invention is contained in or expressed in at least one tissue (for example by modifying the promoter region of a gene according to the invention), or an altered gene product (for example a derivative of the invention is one) Proteins of the SNSR family, for example also a fragment), are contained in or expressed in at least one tissue.
  • tissue for example by modifying the promoter region of a gene according to the invention
  • an altered gene product for example a derivative of the invention is one
  • Proteins of the SNSR family for example also a fragment
  • the native sequences (s) according to the invention can be supplemented with at least one nucleotide sequence according to the invention or be substituted by at least one DNA sequence according to the invention.
  • the substituted and / or supplemented sequence (s) can be sequences according to the invention which are non-native in nature.
  • Transgenic and / or “knock-out” animals in particular mice, rats, pigs, cattle, sheep, fruit flies (Drosophila), C. elegans or zebrafish, are produced in a manner familiar to those skilled in the art in relation to sequences according to the invention expresses a cDNA sequence according to the invention or native or non-native variant in transgenic mice, for example under an NSE promoter in neurons, under an MBP promoter in oligodendrocytes etc.
  • the genetically modified animals can then be examined in different disease models (e.g. experimentally induced stroke, MCAO).
  • all multicellular organisms can be transgenic, in particular mammals, for example mice, rats, sheep, cattle or pigs.
  • transgenic plants are also conceivable.
  • the transgenic animals can also be so-called "knock-out" animals.
  • the transgenic animals can contain a functional or non-functional nucleic acid sequence according to the invention or a functional or non-functional nucleic acid construct alone or in combination with a functional or non-functional sequence which codes for proteins according to the invention.
  • transgenic non-human organisms described above are transgenic animals, in their germ cells or all or part of the somatic cells or in their germ cells or all or part of the somatic cells the native (s) according to the invention ( n) Nucleotide sequence (s) of the SNSR family, in particular the sequences according to FIGS. 3 or 5), have been changed by genetic engineering methods or have been interrupted by inserting DNA elements. Another possibility of using a nucleotide sequence according to the invention or parts thereof is the generation of transgenic or knock-out or conditional or region-specific knock-out animals or specific mutations in genetically modified animals (Ausubel et al.
  • animal models Via transgenic overexpression or genetic mutation (zero mutation or specific deletions, insertions or changes) by homologous recombination in embryonic stem cells, animal models can be generated which provide valuable further information about the (patho-) physiology of the sequences according to the invention. Animal models made in this way can eat- tial test systems for evaluating novel therapeutic agents that affect the biological function of proteins according to the invention, in particular proteins with one of the sequences according to FIGS. 4 and 6, for neural or other processes.
  • Another object of the invention is an antibody which is directed against an epitope on a gene product already described as above, in particular a peptide or protein according to the invention.
  • antibody includes i.S. of the present invention, both polyclonal antibodies and monoclonal antibodies, chimeric antibodies, humanized antibodies, which can all be present in bound or soluble form, and also fragments of the abovementioned antibodies.
  • antibodies according to the invention can also occur in recombinant form as fusion proteins with other (protein) components. Fragments as such or fragments of antibodies according to the invention as constituents of fusion proteins are typically produced by the methods of enzymatic cleavage, protein synthesis or the recombination methods familiar to the person skilled in the art.
  • Antibodies according to the present invention are both polyclonal, monoclonal, human or humanized or recombinant antibodies or fragments thereof, single chain antibodies, for example scFv constructs, or synthetic antibodies.
  • the polyclonal antibodies are heterogeneous mixtures of antibody molecules which are produced from sera from animals which have been immunized with an antigen.
  • the subject of the invention also includes polyclonal monospecific antibodies which are obtained after the antibodies have been purified (for example via a column which is loaded with peptides of a specific epitope).
  • a monoclonal antibody contains an essentially homogeneous population of antibodies that are specifically directed against antigens, the antibodies having essentially the same epitope binding sites. point.
  • Monoclonal antibodies can be obtained by methods known in the art (e.g., Koehler and Milstein, Nature, 256, 495-397, (1975); U.S.
  • Patent 4,376,110 Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring, Harbor Laboratory (1988); Ausubel et al., (Eds), 1998, Current Protocols in Molecular Biology, John Wiley & Sons, New York).
  • the description contained in the abovementioned references is included as part of the present invention in the disclosure of the present invention.
  • Antibodies according to the invention which have been genetically engineered can also be produced by methods as described in the aforementioned publications.
  • antibody-producing cells are grown for this purpose and the mRNA, provided the optical density of the cells is sufficient, is cell-lysed with guanidinium thiocyanate, acidified with sodium acetate, extracted with phenol, chloroform / isoamyl alcohol, precipitated with isopropanol and washed with ethanol from the cells in a known manner Way isolated. Then the reverse transcriptase is used to synthesize cDNA from the mRNA.
  • the synthesized cDNA can be inserted directly or after genetic manipulation, for example by "site directed mutagenesis", introduction of insertions, inversions, deletions or base exchanges into suitable animal, fungal, bacterial or viral vectors and expressed in the corresponding host organisms.
  • Bacterial or yeast vectors such as pBR322, pUC18 / 19, pACYC184, lambda or yeast mu vectors for cloning the genes and expression in bacteria such as E. coli or in yeast such as Saccharomyces cerevisiae are preferred.
  • Antibodies according to the invention can belong to one of the following immunoglobulin classes: IgG, IgM, IgE, IgA, GILD and possibly a subclass of the abovementioned classes, such as the subclasses of the IgG or mixtures thereof.
  • IgG and its subclasses such as, for example, IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgGM are preferred.
  • the IgG subtypes IgG1 / k or IgG2b / k are particularly preferred.
  • a hybridoma cell clone which produces monoclonal antibodies according to the invention can be cultured in vitro, in situ or in vivo.
  • chimeric antibodies according to the invention are molecules which contain different constituents, and these are derived from different animal species (eg antibodies, the variable region which is derived from a mouse monoclonal antibody and a constant one) Region of a human immunoglobulin). Chimeric antibodies are preferably used, on the one hand, to reduce the immunogenicity in use and, on the other hand, to increase the yields in production, for example murine monoclonal antibodies give higher yields from hybridoma cell lines, but also lead to a higher immunogenicity in humans, so that human / murine chimeric antibodies are preferably used.
  • a monoclonal antibody that combines the hypervariable complementarity defining region (CDR) of a murine monoclonal antibody with the remaining areas of a human antibody.
  • CDR complementarity defining region
  • Such an antibody becomes a humanized antibody Chimeric antibodies and methods for their production are known from the prior art (Cabilly et al., Proc. Natl. Sei. USA 81: 3273-3277 (1984); Morrison et al. Proc. Natl. Acad. Sei USA 81: 6851-6855 (1984); Boulianne et al.
  • antibody is intended to include both intact molecules and fragments thereof. Fragments include all shortened or modified antibody fragments with one or two antigen-complementary binding sites, such as antibody parts with a binding site formed by light and heavy chains corresponding to the antibody, such as Fv, Fab or F (ab ') 2 fragments or single-strand fragments. Shortened double-strand fragments such as Fv, Fab or F (ab ') 2 are preferred. Fab and F (ab ') 2 fragments lack an Fc fragment, such as present in an intact antibody, so that they can be transported faster in the bloodstream and have comparatively less non-specific tissue binding than intact antibodies. Such fragments are typically made by proteolytic cleavage using enzymes such as. B. papain (for the production of Fab fragments) or pepsin (for the production of F (ab ') 2, fragments) can be used, or can be obtained by chemical oxidation or by genetic engineering manipulation of the antibody genes.
  • the antibody when it is directed against a peptide or protein for which a nucleic acid or polynucleotide, or a polynucleotide according to FIGS. 3 or 5, at least 90%, preferably 95%, in particular 97%, or a polynucleotide, or a polynucleotide at least 90%, preferably 95%, in particular 97% homologous, which contains the sequence according to FIGS. 3 or 5 or a polynucleotide, or a polynucleotide at least 90%, preferably 95%, in particular 97% homologous , which codes by means of a production method based on a gene fragment according to one of FIGS. 3 or 5.
  • Particularly preferred antibodies of the present invention are directed against a sequence section on the extracellular domain of SNSR-L2 and / or SNSR-L3 as an epitope.
  • Another object of the present invention is an antisense polynucleotide or an antisense nucleic acid, which may also be a PNA which has a sequence which is capable of binding specifically to a polynucleotide according to the invention.
  • PNA peptidic nucleic acid
  • An antisense nucleic acid shows the complementary base sequence to at least part a base nucleic acid.
  • the antisense nucleic acid according to the invention is preferably part of a ribozyme, in particular a hammerhead ribozyme, or a DNA enzyme or another catalytic RNA or DNA.
  • Ribozyme is a catalytically active ribonucleic acid
  • DNA enzyme is a corresponding deoxyribonucleic acid, ie catalytic RNA or DNA.
  • Another embodiment of the antisense nucleic acid according to the invention forms an siRNA which is directed against one of the genes according to the invention.
  • siRNA is a double-stranded RNA molecule (dsRNA) which comprises 19 to 29 bp, in particular 21 to 23 bp, and which has a sequence complementary to the mRNA of the genes according to the invention.
  • dsRNA double-stranded RNA molecule
  • SiRNA molecules can be obtained from various providers, e.g. IBA GmbH (Göttingen, Germany).
  • the siRNA of the present invention is chemically modified, in particular, as further explained below, in order to avoid premature degradation by nucleases.
  • an “antisense polynucleotide” or an “antisense nucleic acid” according to the present invention is thus a molecule consisting of several natural or modified nucleic acid building blocks, the base sequence of which is at least partially or partially complementary to the base sequence of a partial area of a naturally occurring species, For example, the naturally occurring mRNA. Due to the complementarity, the antisense polynucleotide according to the invention or the antisense nucleic acid according to the invention is capable of hybridizing with the target molecule under standard conditions, as defined above, preferably under stringent conditions.
  • the antisense nucleic acid or the antisense polynucleotide comprises DNA or RNA species which contain or can consist of unmodified or modified nucleotides.
  • RNA species such as antisense RNA and siRNA
  • the latter has at least one analogue of naturally occurring nucleotides for stabilization against degradation by RNAsen. This is due to the fact that the cells recognize the RNA-degrading enzyme as a substrate, preferably naturally occurring nucleotides.
  • nucleotide analogs which can be used according to the invention are phosphoramidates, phosphorothioates, peptide nucleotides (ie the antisense nucleic acid is at least partially a peptide nucleic acid (PNA)), methylphosphonates, 7-deazaguaonsine, 5-methylcytosine and inosine cytosine to be named.
  • methods for the expression of SNSR gene products according to the invention are further disclosed, for which host cells are transformed with an expression vector according to the invention.
  • This method for the expression of gene products based on a nucleotide sequence according to the invention does not serve to concentrate and purify the corresponding gene product, but rather to influence the cell metabolism by introducing the nucleotide sequences according to the invention via the expression of the associated gene product.
  • the use of the host cells transformed with the aid of expression vectors is to be considered as a medicament or for the production of a medicament, in particular for the purpose of treating pain, particularly chronic pain.
  • host cells according to the invention are made available in the case of diseases which are based on incorrect regulation of the development and / or transmission of pain.
  • the ex vivo transformed autologous or allogeneic host cells can then be transplanted to patients.
  • a method for isolating gene products according to the invention (preferably with at least one with the amino acid sequences according to the invention), in particular the sequences according to FIGS. 4 or 6, is at least via a partial sequence of at least 10, preferably 20, more preferably at least 30 amino acid homologous partial sequence, the (i) host cells being transformed with an expression vector according to the invention and then being cultivated under suitable conditions which promote expression, optionally under selection pressure (cf. the explanations relating to the identification method according to the invention), so that (ii) the gene product can finally be purified from the culture.
  • the gene product according to the invention of the nucleotide sequence according to the invention can, depending on the expression system, be isolated from a culture medium or from cell extracts.
  • the person skilled in the art can readily recognize that the respective isolation methods and the method for the purification of the recombinant protein encoded by a DNA according to the invention strongly depend on the type of the host cell or also on the fact whether the protein is secreted into the medium.
  • expression systems can be used which lead to the secretion of the recombinant protein from the host cell.
  • the culture medium must be passed through commercially available protein concentration filters, e.g. Amicon or Millipore Pelicon, to be concentrated.
  • a cleaning step can take place, e.g. a gel filtration step or purification using column chromatographic methods.
  • an anion exchanger can be used which has a matrix with DEAE.
  • encoded polypeptide can then serve HPLC steps. It can be one or more steps. In particular, the "reversed phase" method is used. These steps serve to obtain an essentially homogeneous recombinant protein of a nucleotide sequence according to the invention.
  • transformed yeast cells can also be used.
  • the translated protein can be secreted, so that protein purification is simplified.
  • Secreted recombinant protein from a yeast host cell can be obtained by methods as described in Urdal et al. (J. Chromato. 296: 171 (1994)) and form part of the disclosure of the present invention.
  • Nucleic acids according to the invention in particular DNA according to the invention, and / or gene products according to the invention can be used as medicaments or for the production of a medicament. These can be administered as such (for example, buccally, intravenously, orally, parenterally, nasally, subcutaneously) or in combination with other active ingredients, auxiliaries or additives typical of drugs.
  • Nucleic acid according to the invention can be injected as a naked nucleic acid, in particular intravenously, or can be administered to the patient using vectors. These vectors can be plasmids as such, but also viral vectors, in particular retroviral or adenoviral vectors, or also liposomes which can have naked DNA according to the invention or a plasmid which contains DNA according to the invention.
  • sequences according to the invention in particular the nucleotide or amino acid sequences of the figures mentioned or their variants, and protein heteromers according to the invention and reagents according to the invention derived therefrom (oligonucleotides, antibodies, peptides) is thus used for the production of a medicament for therapeutic purposes, ie Treatment of diseases.
  • the therapeutic use for the treatment or for the manufacture of a medicament for the treatment of pain, in particular diseases or pathophysiological is very particularly preferred.
  • logical states that are based on the misguided development and / or transmission of pain, preferably of a chronic nature.
  • nucleic acids according to the invention for example nucleic acids according to the invention, antisense nucleic acids, oligo- or polypeptides, expression vectors, host cells or surrogate ligands, which can attach to all positions of the receptors according to the invention relevant for regulation, in particular for the production of medicaments Treatment for pain into consideration.
  • cell-based HTS tests according to the invention prove to be suitable for functional receptor activation, measured by enzyme complementation.
  • the test is based on the general regulatory mechanism of GPCRs and measures the interaction between the activated receptor and ß-arrestin.
  • inactive complementary ß-galactosidase fragments are fused to the C-terminus of the receptor and to ß-arrestin.
  • Activating the receptor recruits beta-arrestin.
  • the two halves of the beta-galactosidase are brought together so that a functioning beta-galactosidase enzyme is formed, which can convert the corresponding substrate and serve as a measurement signal (ICAST system).
  • ICAST system measurement signal
  • this test can be carried out with all enzymes which can be expressed as fusion proteins by two complementary halves and which carry out a substrate reaction which can be detected by conventional measuring methods.
  • Another object of the present invention is related to the therapeutic use of sequences according to the invention, namely the use of a nucleic acid sequence or protein sequence according to the invention, including all derivatives, alleles, fragments etc., as defined above, for gene therapy in mammals, for example in humans or else such gene therapy procedures.
  • Gene therapy encompasses all forms of therapy that either nucleotide sequences according to the invention in the body or parts thereof, For example, introduce individual tissues or influence the expression of sequences according to the invention.
  • all modifications familiar to the person skilled in the art can be used in the context of gene therapy, for example oligonucleotides, for example antisense or hybrid RNA-DNA oligonucleotides, with any modifications which contain sequences according to the invention.
  • Viral constructs containing a sequence according to the invention can also be used.
  • Corresponding naked DNA sequences according to the invention can also be considered in the context of gene therapy.
  • Nucleic acid pieces with enzymatic activity eg ribozymes
  • nucleic acids or polypeptides according to the invention protein heteromers according to the invention and reagents according to the invention (oligonucleotides, antibodies, peptides) derived therefrom are also possible, for example for the diagnosis of human diseases or genetic predispositions, for example also in the context of pregnancy examinations. These diseases or predispositions are, in particular, the diseases mentioned above in connection with therapeutic applications, especially in connection with pain.
  • diagnostic methods can be designed as in vivo, but typically ex vivo methods. A typical application of a diagnostic method according to the invention for the qualitative and / or quantitative detection of a nucleic acid according to the invention in a biological sample will serve ex vivo.
  • Such a method preferably comprises the following steps: (A) incubation of a biological sample with a known amount of nucleic acid according to the invention or a known amount of oligonucleotides which are suitable as primers for an amplification of the nucleic acid according to the invention, (B) detection of the nucleic acid according to the invention by specific hybridization or PCR amplification, (C) comparison the amount of hybridizing nucleic acid or of nucleic acid obtained by PCR amplification with an amount standard.
  • Sequences according to the invention can be used in methods for the determination of their polymorphisms e.g. used in humans. These determined polymorphisms of sequences according to the invention are not only subject to the disclosure of the present invention, but can also be used as prognostic markers for the diagnosis or for the diagnosis of a predisposition to diseases which are associated with a sequence by infunctional expression according to the invention, expression of infunctional sequences according to the invention and / or serve their overexpression. In addition, sequences according to the invention allow research into human hereditary diseases, both monogenic and polygenic diseases.
  • nucleic acids or polypeptides according to the invention are also suitable for scientific use.
  • sequences according to the invention allow, in a manner known to those skilled in the art, for example to identify sequences related to single-cell or multicellular organisms via cDNA libraries or to localize sequences related to the human genome.
  • the nucleotide sequences according to the invention in particular the sequences according to FIGS.
  • 3 and 5 can thus be used to generate genes for mRNAs which code for these nucleic acids or their functional equivalents, homologs or derivatives, for example murine or others
  • genes for mRNAs which code for these nucleic acids or their functional equivalents, homologs or derivatives, for example murine or others
  • nucleic acids With the aid of nucleic acids according to the invention, it is possible in particular to diagnose human hereditary diseases, both monogenic and polygenic diseases, which is why they are used as markers, which results in a diagnostic method according to the invention for hereditary diseases.
  • a test system is disclosed according to the invention for scientific use, which is based on amino acid and / or nucleotide sequences according to the invention.
  • the cDNA, the genetic DNA, the regulatory elements of the nucleic acid sequences according to the invention, and also the polypeptide, and partial fragments thereof can be used in recombinant or non-recombinant form for the development of a test system.
  • test system is particularly suitable for measuring the activity of the promoter or of the protein in the presence of the test substance.
  • test substances are preferably simple measurement methods (colorimetric, luminometric, based on fluorescence or radioactive), which allow the rapid measurement of a large number of test substances (Böhm, Klebe, Kubyi, 1996, active ingredient design, Spektrum Verlag, Heidelberg) .
  • the test systems described allow chemical libraries to be searched for substances which have inhibitory or activating effects on proteins according to the invention, in particular the sequences according to FIGS. 4 and 6 (or their derivatives or fragments). The identification of such substances is the first step on the way to identifying new drugs that specifically affect SNSR-associated signal transduction.
  • test systems are provided which use the known properties of G-protein-coupled proteins, for example the test systems disclosed below.
  • ribozymes are used which can cut a target mRNA.
  • ribozymes are therefore disclosed and are able to cleave an object of the present invention native SNSR mRNA, for example from SNSR-L2 or SNSR-L3.
  • Ribozymes according to the invention must be able to interact with the target mRNA according to the invention, for example via base pairing, and then cleave the mRNA in order to block the translation of, for example, SNSR-L2 or SNSR-L3.
  • the ribozymes according to the invention are introduced into the target cells via suitable vectors (in particular plasmids, modified animal viruses, in particular retroviruses), the vectors, in addition to possibly other sequences, having a cDNA sequence for a ribozyme according to the invention).
  • suitable vectors in particular plasmids, modified animal viruses, in particular retroviruses
  • the vectors in addition to possibly other sequences, having a cDNA sequence for a ribozyme according to the invention.
  • a chemical compound according to the invention will modulate, typically inhibit or also activate, the intracellular and / or extracellular function of the proteins of the SNSR family according to the invention, or at the level of the underlying DNA sequences according to the invention, for example by binding to the DNA (for example the Promoter region) or by binding to one of the transcription factors controlling a gene according to the invention, which influence biological function.
  • Compounds according to the invention will typically bind specifically to a protein according to the invention, in particular to a protein with one of the amino acid sequences according to FIGS. 4 or 6, or to a nucleic acid sequence according to the invention, in particular to a nucleic acid with a nucleotide sequence according to one of FIGS. 3 or 5, and thereby produce a pharmacological, in particular analgesic effect.
  • Compounds according to the invention are advantageously obtainable by the screening method defined above.
  • Chemical compounds are therefore disclosed according to the invention, preferably an organic chemical compound with a molecular weight of ⁇ 5000, in particular ⁇ 3000, in particular ⁇ 1500, which is typically physiologically well tolerated and can preferably pass through the blood-brain barrier. Possibly. it will be part of a composition with at least one further active ingredient and preferably auxiliaries and / or additives and can be used as a medicament.
  • the organic molecule will be particularly preferred if the binding constant for binding to an inventive protein, in particular to the cytosolic domain or to the extracellular domain of a protein according to the invention, is at least 10 7 mol "1.
  • the compound according to the invention will preferably be such that it can pass through the cell membrane, either by diffusion or via (intra) Membrane transport professionals, if appropriate after appropriate modification, for example with a coupled amino acid sequence, further preferred are those compounds which inhibit or intensify the interaction of proteins of the SNSR family according to the invention with binding partners, in particular for the transduction of a pain signal Positions on the surface of proteins according to the invention or cause a local conformational change in the proteins according to the invention, so that the binding of a native binding partner to a protein according to the invention is prevented.
  • Structural analyzes of a protein according to the invention specifically allow compounds according to the invention to be found which have a specific binding affinity (rational drug design (Böhm, Klebe, Kubinyi, 1996, drug design, Spektrum Verlag, Heidelberg)).
  • a specific binding affinity rational drug design (Böhm, Klebe, Kubinyi, 1996, drug design, Spektrum Verlag, Heidelberg)
  • the structure or a partial structure, derivative, allele, isoform or part of one of one of the proteins according to the invention in particular of one of the proteins with the sequences according to one of FIGS.
  • the compound according to the invention is an antibody, preferably an antibody directed against a protein of the SNSR family according to the invention, for example SNSR-L2 or SNSR-L3, or also an antibody directed against the underlying mRNA
  • the ex vivo is introduced into retransplanted host cells or by gene therapy in vivo processes into host cells and is not secreted there as an "intrabody”, but rather can exert its effect intracellularly.
  • the cells can be protected against a misdirected pain reaction, for example by overexpression of a protein according to the invention.
  • a connection according to the invention with the function of the blockade, but possibly also activation of the biological function of native SNSR protein according to the invention, e.g. of sequences according to FIGS. 4 and 6, or corresponding native alleles or native splice variants, e.g. the pain-relevant function, can find use as a medicinal product.
  • all of the aforementioned variants are included as compounds, for example, organic chemical compounds, antibodies, anti-sense oligonucleotides, (hammerhead) ribozymes, siRNA.
  • An inhibitor according to the invention for example an inhibitory antibody (in particular an intrabody), a (hammerhead) ribozyme, anti-sense RNA, in particular siRNA, dominant-negative mutants or one of the aforementioned possibly inhibitory organic chemical compounds, preferably a high-affinity compound, obtainable, for example, from one of the abovementioned methods) of the cellular function of a native protein according to the invention, in particular a protein with the sequences according to FIGS. 4 and 6, or of its native variants, for example the chronic pain reaction, as a medicament and very particularly in the treatment of chronic pain can be used.
  • an inhibitory antibody in particular an intrabody
  • a (hammerhead) ribozyme anti-sense RNA, in particular siRNA, dominant-negative mutants or one of the aforementioned possibly inhibitory organic chemical compounds, preferably a high-affinity compound, obtainable, for example, from one of the abovementioned methods
  • a native protein according to the invention in particular a protein with the sequences
  • the invention further relates to a medicament comprising at least one nucleic acid according to the invention, an antisense nucleic acid according to the invention, a peptide or protein according to the invention, a vector according to the invention, an antibody according to the invention, a host cell according to the invention and / or a compound according to the invention and optionally suitable auxiliaries, Carriers and / or additives.
  • the medicament according to the invention is preferably used to treat pain, in particular of a chronic nature, which can be neuropathic and / or inflammation-related, for example.
  • auxiliary and additives for parenteral administration include, for example, sterile water, sterile saline, polyalkylene glycols, hydrogenated naphthalenes and in particular biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers.
  • compositions according to the invention can contain filling substances or substances such as lactose, manitol, substances for covalently attaching polymers such as polyethylene glycol to invented nucleic acids according to the invention, proteins or antibodies, complexation with metal ions or inclusion of materials in or on special preparations of polymer compounds, such as polylactate, polyglycolic acid, hydrogel or on liposomes, microemulsions, micelles, unilameric or multilamelar vesicles, erythrocyte fragments or spheroblasts.
  • polylactate polyglycolic acid
  • hydrogel or on liposomes such as polylactate, polyglycolic acid, hydrogel or on liposomes, microemulsions, micelles, unilameric or multilamelar vesicles, erythrocyte fragments or spheroblasts.
  • the respective embodiments of the pharmaceutical compositions are selected depending on the physical behavior, for example with regard to solubility, stability, bioavail
  • Controlled or constant release of the active ingredient components according to the invention includes the formulation based on lipophilic depots (eg fatty acids, waxes or oils).
  • lipophilic depots eg fatty acids, waxes or oils.
  • coatings of pharmaceutical compositions or pharmaceuticals according to the invention containing the therapeutically active substances namely coatings with polymers (for example polyoxamers or polyoxamines) are also disclosed.
  • therapeutically active substances or compositions according to the invention can have protective coatings, for example protease inhibitors, nuclease inhibitors or permeability enhancers.
  • Preferred carriers are typically aqueous carrier materials, using water for injection (WFI) or water buffered with phosphate, citrate, HEPES or acetate, etc.
  • the carrier or vehicle will preferably also contain salt components, for example sodium chloride, potassium chloride or other components which make the solution, for example, isotonic.
  • the carrier or vehicle can furthermore contain additional components, such as human serum albumin (HSA), polysorbate 80, sugar or amino acids, etc.
  • the medicaments according to the invention can be administered as liquid medicinal forms in the form of injection solutions, drops or juices, as semi-solid medicinal forms in the form of granules, tablets, pellets, patches, capsules, plasters or aerosols and, depending on the galenic form, may contain them in addition to the at least one object according to the invention
  • carrier materials fillers, solvents, diluents, dyes and / or binders.
  • excipients and the amounts to be used depends on whether the medicinal product is oral, oral, parenteral, intravenous, intraperitoneal, intradermal, intramuscular, intranasal, buccal, rectal or local, for example on infections on the skin, mucous membranes and on the eyes to be applied.
  • Preparations in the form of tablets, dragees, capsules, granules, drops, juices and syrups are suitable for oral administration, and solutions, suspensions, easily reconstitutable dry preparations and sprays are suitable for parenteral, topical and inhalation administration.
  • Objects according to the invention in a depot in dissolved form or in a plaster, optionally with the addition of agents which promote skin penetration, are suitable percutaneous application preparations.
  • Forms of preparation which can be used orally or percutaneously can release the articles according to the invention with a delay.
  • the amount of active ingredient to be administered to the patient varies depending on the weight of the patient, the type of application, the indication and the severity of the disease. 2 to 500 mg / kg of at least one object according to the invention are usually applied. If the medicament is to be used in particular for gene therapy, physiological saline, stabilizers, proteinase, DNAse inhibitors, RNAse inhibitors etc. are recommended as suitable auxiliaries or additives.
  • a drug is particularly preferred which contains the preferred forms listed above or the particularly selected form (s) of the nucleic acids), the antisense nucleic acid (s), gene products (s), in particular the above peptides or proteins, vectors, antibodies, host cells , and / or contains connections.
  • the present invention provides a diagnostic agent containing at least one nucleic acid according to the invention, an antisense nucleic acid according to the invention, a gene product according to the invention, in particular a peptide or protein according to the invention, a vector, antibodies or parts thereof and / or a host cell according to the invention and, if appropriate, suitable auxiliaries and / or additives. Diagnostic means an aid for diagnosing, for example, an illness associated with pain.
  • a diagnostic agent which contains the above-preferred form of the nucleic acids, antisense nucleic acid (s), gene products, in particular peptides or proteins or a part thereof, vectors, antibodies and / or the host cell.
  • a form of the diagnostic agent is particularly preferred which contains an antisense nucleic acid which has a sequence which is capable of specifically binding to a nucleic acid according to the invention.
  • diagnostic in vitro methods are disclosed according to the invention, which make it possible to investigate in an organism, in particular in humans, on a molecular basis the cause of pathological pain conditions that occur, or to demonstrate illnesses associated with pain (cf. also the preceding explanations on the various possible uses of objects according to the invention) ).
  • PCR methods for example RT-PCR methods, ie the diagnosis based on mRNA, which are correspondingly translated into cDNA in vitro and are then reproduced using conventional PCR methods, are particularly suitable for such uses.
  • Corresponding array techniques which position oligonucleotides according to the invention on a chip, also allow diagnostics with the aid of hybridization reactions.
  • the patient sample is tested against an array with oligonucleotides according to the invention, which represent the sequences according to the invention.
  • Corresponding signals on the array in the case of oligonucleotides which differ from a comparison experiment with a sample of a healthy organism therefore permit a corresponding diagnosis.
  • Such diagnoses at the nucleic acid level can of course also be carried out using classic blot methods (Northern and / or Southern blot).
  • Tests with (antibodies) against the amino acid sequences according to the invention are also suitable in a similar manner in order to change the expression of the proteins or peptides associated with the pain regulation according to the invention in a sick organism, ie an organism which is burdened with acute, preferably chronic pain, in particular a human patient , against a healthy comparative organism.
  • Appropriate test formats for example radioimmunoassays, ELISA tests, etc., are available to a person skilled in the art.
  • Another object of the invention is the use of an object according to the invention, for example a nucleic acid, an antisense nucleic acid, a gene product, in particular a peptide or protein, vector, antibody, a host cell and / or a compound for producing a medicament for the treatment of pain or as a medicine or for use as a medicine for the treatment of pain.
  • an object according to the invention for example a nucleic acid, an antisense nucleic acid, a gene product, in particular a peptide or protein, vector, antibody, a host cell and / or a compound for producing a medicament for the treatment of pain or as a medicine or for use as a medicine for the treatment of pain.
  • neuropathic or inflammation-related pain is particularly preferred.
  • Figure 1 shows the genomic organization of the three rat SNSR genes.
  • the distance between the SNSR-L3 and the SNSR-L2 genes is approximately 100 kB, while the distance between the SNSR-L2 gene and the SNSR gene is approximately 48 kB.
  • the orientation is indicated with an arrow for each gene.
  • Figure 2 shows the genomic sequence of the rat SNSR gene cluster.
  • the SNSR gene includes positions 179475 to 180485 (or 180488 including stop codon).
  • the SNSR-L2 gene included positions 132249 to 133316 (or 133319 including stop codon)
  • the SNSR-L3 gene is inversely oriented and comprises positions 33328 to 32417 (or 32414 including stop codon).
  • FIG. 3 shows the nucleotide sequence of the rat SNSR-L2 gene (nucleotides 250 to 131 (or 1320 including stop codon)).
  • Figure 4 shows the amino acid sequence of the rat SNSR-L2 receptor.
  • FIG. 5 shows the nucleotide sequence of the rat SNSR-L3 gene (nucleotides 1 to 912 (or 915 including stop codon)).
  • Figure 6 shows the amino acid sequence of the rat SNSR-L3 receptor.
  • FIG. 7 shows a dendrogram to show the structure of the SNSR gene family.
  • Step Example. 1 Identification of new SNSR genes in the rat oenome
  • rat genomic data from publicly funded genome projects, as stored in the "HTGS" section of the EMBL / GenBank database, were searched for sequences with similarity to human SNSR proteins with the help of the BLAST program. The candidates found during the search were then aligned with the "ClustalW” program and subjected to a dendrogram analysis (see below) to determine whether they were members of the internal SNSR family.
  • the only known SNSR gene of the rat is intronless. According to the invention, however, it was found that it lies in a genomic cluster of several mutually related genes.
  • a genomic sequence FIG. 2 which contains the known SNSR gene of the rat (positions 179475 to 180485 (or 180488 including stop codon) of the sequence according to FIG. 2)
  • two further SNSR genes (SNSR- L2 and SNSR-L3) identified.
  • FIG. 1 shows the genomic organization of the SNSR genes of the rat.
  • Figure 2 shows the sequence of the rat SNSR cluster.
  • FIG. 3 shows the cDNA sequence of the newly identified receptor SNRS-L2 and FIG. 4 the amino acid sequence derived therefrom.
  • FIG. 5 shows the cDNA sequence of the further identified receptor SNRS-L3 and FIG. 6 the amino acid sequence derived therefrom.
  • Example 3 Example of a medicament containing an active ingredient according to the invention - tablet formulation
  • Tablets can be produced by directly compressing mixtures of the active ingredient according to the invention with corresponding auxiliaries or by compressing granules containing the active ingredient (with optionally other auxiliaries).
  • the granules can either by wet granulation with e.g. aqueous granulating liquids and subsequent drying of these granules or by dry granulation e.g. be made via compacting.
  • LudipressTM granules for direct tableting from lactose monohydrate, povidone K30 and crospovidone
  • Example 4 Example of a medicament containing an inert substance according to the invention
  • 1 g of an active ingredient according to the invention is dissolved in 1 liter of water for injections at room temperature and then adjusted to isotonic conditions by adding NaCl (sodium chloride).

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Abstract

L'invention concerne des gènes de récepteurs de la famille des SNSR (récepteur spécifique des neurones sensoriels), qui sont impliqués dans des états pathologiques douloureux, en particulier des douleurs chroniques. L'invention concerne en particulier un procédé pour identifier des composés modulant la fonction des nouveaux récepteurs SNSR, ce qui permet de détecter les substances impliquées dans la douleur. L'invention concerne, en outre, l'utilisation desdits composés ainsi que les acides nucléiques anticorps et antisens utilisés contre les nouveaux récepteurs de la famille des SNSR dans des médicaments. L'invention concerne également des diagnostics pour traiter ou diagnostiquer des douleurs ou des états pathologiques associés.
PCT/EP2004/002384 2003-03-11 2004-03-08 Identification de genes impliques dans la douleur de la famille des snsr (recepteur specifique des neurones sensoriels) WO2004081042A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10310758.4 2003-03-11
DE10310758A DE10310758A1 (de) 2003-03-11 2003-03-11 Identifizierung von schmerzrelevanten Genen der SNSR (Sensory Neuron Specific Receptor)-Familie

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WO2004081042A2 true WO2004081042A2 (fr) 2004-09-23
WO2004081042A3 WO2004081042A3 (fr) 2005-04-21

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001074902A2 (fr) * 2000-04-04 2001-10-11 Janssen Pharmaceutica N.V. Recepteur couple aux proteines g
WO2001083555A2 (fr) * 2000-05-04 2001-11-08 California Institute Of Technology Molecules de signalisation de la douleur

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001074902A2 (fr) * 2000-04-04 2001-10-11 Janssen Pharmaceutica N.V. Recepteur couple aux proteines g
WO2001083555A2 (fr) * 2000-05-04 2001-11-08 California Institute Of Technology Molecules de signalisation de la douleur

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BENDER ECKHARD ET AL: "Characterization of an orphan G protein-coupled receptor localized in the dorsal root ganglia reveals adenine as a signaling molecule." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 25 JUN 2002, Bd. 99, Nr. 13, 25. Juni 2002 (2002-06-25), Seiten 8573-8578, XP002296469 ISSN: 0027-8424 *
DATABASE EMBL 7. April 1999 (1999-04-07), "UI-R-GO-ui-d-12-0-UI.s2 UI-R-GO Rattus norvegicus cDNA CLONE , mRNA SEQUENCE" XP002287773 gefunden im EBI Database accession no. AI578247 *
DONG X ET AL: "A diverse family of GPCRs expressed in specific subsets of nociceptive sensory neurons." CELL. 7 SEP 2001, Bd. 106, Nr. 5, 7. September 2001 (2001-09-07), Seiten 619-632, XP002287771 ISSN: 0092-8674 in der Anmeldung erw{hnt *
LEMBO PAOLA M C ET AL: "Proenkephalin A gene products activate a new family of sensory neuron--specific GPCRs." NATURE NEUROSCIENCE. MAR 2002, Bd. 5, Nr. 3, M{rz 2002 (2002-03), Seiten 201-209, XP002264104 ISSN: 1097-6256 in der Anmeldung erw{hnt *
ZYLKA MARK J ET AL: "Atypical expansion in mice of the sensory neuron-specific Mrg G protein-coupled receptor family." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 19 AUG 2003, Bd. 100, Nr. 17, 19. August 2003 (2003-08-19), Seiten 10043-10048, XP002287772 ISSN: 0027-8424 *

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WO2004081042A3 (fr) 2005-04-21
DE10310758A1 (de) 2004-09-23

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