EP1315751A2 - Deux recepteurs d'un sterol activateur de meiose designes sam1a et sam1b - Google Patents

Deux recepteurs d'un sterol activateur de meiose designes sam1a et sam1b

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Publication number
EP1315751A2
EP1315751A2 EP01957793A EP01957793A EP1315751A2 EP 1315751 A2 EP1315751 A2 EP 1315751A2 EP 01957793 A EP01957793 A EP 01957793A EP 01957793 A EP01957793 A EP 01957793A EP 1315751 A2 EP1315751 A2 EP 1315751A2
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EP
European Patent Office
Prior art keywords
mas
receptor
bind
seq
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01957793A
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German (de)
English (en)
Inventor
Philip Wahl
Henrik Vissing
Christian Grondahl
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer Pharma AG
Novo Nordisk AS
Original Assignee
Schering AG
Novo Nordisk AS
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Filing date
Publication date
Priority claimed from PCT/DK2001/000550 external-priority patent/WO2002016432A2/fr
Application filed by Schering AG, Novo Nordisk AS filed Critical Schering AG
Publication of EP1315751A2 publication Critical patent/EP1315751A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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

Definitions

  • the present invention relates to receptors or signalling proteins of FF-MAS, polynucleotides coding for receptors or signalling proteins of FF-MAS, probes hybridising with nucleic acids encoding receptors or signalling proteins of FF-MAS, DNA constructs comprising a sequence encoding receptors or signalling proteins of FF-MAS, culture cell lines wherein the DNA sequence encodes receptors or signalling proteins of FF-MAS, antibodies specifically binding to receptors or signalling proteins of FF-MAS, hybridoma producing monoclonal antibodies specifically binding to receptors or signalling proteins of FF-MAS, and methods for detecting the presence of a compound having affinity to receptors or signalling proteins of FF-MAS.
  • IVF of human oocytes has become commonly used for the treatment of female and male sub fertility.
  • the standard IVF treatment includes a long phase of hormone stimulation of the female patient.
  • the aspirated oocyte is subsequently fertilised in vitro and cultured. Continuous efforts have been made to optimise and simplify this procedure. Nevertheless, the overall pregnancy rate cannot be increased significantly over about 20% with the current treatment modalities.
  • a large European survey of IVF patients it was found that 7.2 oocytes out of 11.5 aspirated oocytes per patient had undergone resumption of meiosis immediately before fertilisation, only 4.3 oocytes were fertilised and only 2.2 oocytes reached the 8-cell embryo stage after fertilisation and in vitro culture (ESHRE, Edinburgh, 1997).
  • FF-MAS 4,4-dimethyl-5 ⁇ -cholesta-8,14,24-triene-3 ⁇ -ol
  • Receptors are defined as proteinaceous macromolecules that perform a signal transducing function upon ligand binding. Many receptors are located on the outer cell membrane, others are located intracellularly. The substance which is bound by the receptor is called a ligand, a term which is definitionally meaningful only in terms of its counterpart receptor.
  • the term "ligand” does not imply any particular molecular size or other structural or compositional feature other than that the substance in question is capable of binding, cleaving or otherwise interacting with the receptor in such a way that the receptor conveys information about the presence of the ligand to a target molecule. Stated alternatively, not all substances capable of binding a receptor are ligands, but all ligands are capable of binding a receptor. Receptors do not include such substances as immunoglobulins.
  • Receptors are believed to function by a process variously termed activation or signal transduction.
  • a ligand binds to the ligand binding domain in such a way that the conformation of the receptor molecule changes.
  • This conformational change modifies the effect of the receptor on cytoplasmic components.
  • changes brought about by receptor activation are changes in or development of receptor enzymatic activity.
  • Signalling proteins such as cAMP, IP3, kinases, and phosphatases are proteins ubiquitously found in all tissues. These proteins cascade by various pathways, the stimulus from ligand/receptor interaction down stream to cellular events, typically changing the enzymatic activity or functional state of effector molecules.
  • Cytoplasmic proteins can act as receptors or signalling molecules in cascading the stimulus from the ligand to cellular events.
  • Various receptors or signalling protein types make use of different path ways (for example small G proteins, calcium fluxes, phospatases, and lipases), all of them resulting in changes of enzymatic activity or gene transcription.
  • Meiotic activating sterols constitute active signalling molecules first identified in follicular fluid and in bull testicular tissue.
  • the sterols are described by By- skov 1995 and Gr ⁇ ndahl et al. (Biol. Reprod. 58 (1998), 1297 et seq.) and in WO 96/00235, 96/27658, 97/00884, 98/28323, 98/54965 and 98/55498, more specifically in Claim 1 thereof, as being potent activators of the meiotic process.
  • No receptors or signalling proteins have been described to directly or indirectly signalling the meiotic effect of MAS sterols.
  • nucleotide and amino acid sequence of clone NT2RM2001632 was released with accession number AK022554 and on 10 May 2001 , the nucleotide and amino acid sequence of clone NT2RP2000448 was submitted with accession number AK027535. No utility or action was mentioned for these clones.
  • the present invention provides the nucleotide sequence of the receptors or signalling proteins of meiotic acting sterols (MAS).
  • MAS meiotic acting sterols
  • the present invention provides isolated and substantially pure MAS receptors or MAS signalling proteins and fragments thereof. These re- ceptors or signalling proteins have been shown to be involved in the gamete maturation process induced by 3 ⁇ -hydroxy-4,4-dimethylcholest-8,14,24-triene (hereinafter designated FF-MAS), specifically inducing, upon ligand activation, germinal vesicle breakdown (hereinafter designated GVB) in mouse oocyte cultured in vitro.
  • FF-MAS 3 ⁇ -hydroxy-4,4-dimethylcholest-8,14,24-triene
  • a MAS receptor or a MAS signalling protein is defined as a proteinaceous macromolecule that perform a signal trans- ducing function upon binding to FF-MAS.
  • a MAS receptor or a MAS signalling protein binds to FF-MAS.
  • a MAS signalling protein can be designated a MAS binding protein.
  • a MAS receptor is any protein related to the protein SAM1a or SAMlb that possess the same functional characteristic regarding the interaction with FF-MAS or other endogenous meiosis activating sterols, for example, 3 ⁇ -hydroxycholest-8,14-diene; 3 ⁇ -hydroxy-4,4- dimethylcholest-8,24-diene; and 3 ⁇ -hydroxycholest-8,24-diene, or their metabolites (as ligand).
  • Functional characteristics include binding, receptor activation, and subsequent germinal vesicles breakdown (GVB) in oocytes.
  • GVB germinal vesicles breakdown
  • the MAS receptor can be used to discover profertility and antifertility compounds which can be used to men and women.
  • the invention also provides antibodies to the MAS receptor or signalling protein, in the form of antisera and/or monoclonal antibodies.
  • the invention provides the ability to produce the MAS receptor or MAS signalling protein and polypeptides or fragments thereof by recombinant means.
  • the expressed MAS receptor or signalling protein or fragments may or may not have the biological activity of native receptor or signalling protein.
  • isolated and purified polynucleotides are described which code for the receptor or signalling protein and fragment thereof, where the polynucleotides may be in the form of DNA, such as cDNA, or RNA. Based on these sequences, probes may be used to hybridise and identify these and related genes which encode MAS receptors or MAS signalling proteins.
  • the probes may be full length cDNA or as small as form 14 to 25 nucleotide, more often though from about 40 to about 50 or more nucleotides.
  • the invention concerns DNA constructs which comprise a transcriptional promoter, a DNA sequence which encodes the receptor or signalling protein or fragment, and a transcriptional terminator, each operably linked for expression of the receptor or signalling protein.
  • the construct may also contain at least one signal se- quence.
  • the expressed receptor or signalling protein may also be isolated from the cells by, for example, immunoaffinity purification.
  • Cells or bacteria which express the MAS receptor or MAS signalling proteins may also be used to identify compounds which can alter the receptor or signalling protein-mediated me- tabolism of a cell.
  • Compounds may be screened for binding to the receptor or signalling protein, and/or for effecting a change in receptor or signalling protein-mediated metabolism in the host cell.
  • Agonists and/or antagonists of the MAS receptor or MAS signalling proteins may also be screened in cell-free systems using purified receptor or signalling proteins or binding fragments thereof for the effect on ligand/receptor interaction or ligand/signalling protein interaction, or using reconstituted systems such as micelles which also provide the ability to assess metabolic changes.
  • the invention relates to methods for diagnosis, where the presence of a mammalian MAS receptor or MAS signalling protein in a biological sample may be determined.
  • a monospecific antibody which specifically binds the receptor or signalling protein is incubated with the sample under conditions conducive to immune complex formation, which complexes are then detected, typically by means of a label such as an enzyme, fluorophore, radionuclide, chemiluminiscer, particle, or a second labelled antibody.
  • a label such as an enzyme, fluorophore, radionuclide, chemiluminiscer, particle, or a second labelled antibody.
  • the receptor proteins or signalling proteins of this invention can be said to belong to a novel super family of oxysterol binding proteins (hereinafter designated OSPB) recently published in J.Lipid.Res. 40 (1999), 2204. No function whatsoever in gamete maturation of either gender or regulation of any meiotic processes has been assigned to this OSPB family.
  • OSPB novel super family of oxysterol binding proteins
  • SEQ ID NO: 1 and SEQ ID NO: 3 are the nucleotides of the cDNA from two mouse MAS receptors or signalling peptides, designated SAM1a and SAM 1b, respectively, and having the amino acid sequences stated in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.
  • SEQ ID NO: 5 and SEQ ID NO: 7 are the nucleotides of the cDNA from two human MAS receptors or signalling peptides, designated SAM1a and SAM 1b, respectively, and having the amino acid sequences stated in SEQ ID NO: 6 and SEQ ID NO: 8, respectively.
  • SEQ ID NO: 9 through 14 are the nucleotides referred to in example 5.
  • the present invention presents the means to identify agonists, and antagonists of the MAS receptor/ligand interaction or MAS signalling protein/ligand interaction by providing isolated MAS receptor or MAS signalling protein.
  • MAS receptor refers to any proteins either derived from a naturally occurring MAS receptor, or which shares significant structural and functional characteristics peculiar to a naturally occurring MAS receptor. Such a receptor may result when regions of a naturally occurring receptor are deleted or replaced in such a manner as to yield a protein having a similar function.
  • homologous sequences allelic variations, and natural mutants; induced point, deletion, and insertion mutants; alternatively expressed variants; proteins encoded by DNA which hybridise under high or low stringency conditions to nucleic acids which encode naturally occurring MAS receptor; proteins retrieved from naturally occurring materials; and closely related proteins retrieved by antisera directed against MAS receptor proteins are also included. Similarly, this applies to MAS signalling proteins.
  • MAS receptor is meant a molecule capable of being bound by the ligand-binding domain of MAS receptor, a MAS receptor analogue, or chimeric MAS receptor as generally described in US Patent specification No. 4,859,609, incorporated by reference herein.
  • the molecule may be chemically synthesised or may occur in nature.
  • Ligands may be grouped into agonists and antagonists. Agonists are those molecules whose binding to a receptor induces the response pathway within a cell. Antagonists are those molecules whose binding to a receptor blocks the response pathway within a cell.
  • isolated MAS receptor or MAS signalling protein refers to MAS receptor or MAS signalling protein which is in other than its native environment such as a mammalian oocyte, including, for example, substantially pure MAS receptor as defined herein below. More generally, isolated is meant to include MAS receptor or MAS signalling protein as a heterologous component of a cell or other system.
  • MAS receptor or MAS signalling protein may be expressed by a cell transfected with a DNA construct which encodes MAS receptor or MAS signalling protein, separated from the cell and added to micelles which contain other selected receptor or signalling proteins.
  • the term MAS re- ceptor covers both a MAS receptor and a MAS signalling protein.
  • purified MAS receptor or MAS signalling protein is meant MAS receptor or MAS signalling protein having a purity of at least 50%, preferably at least 80%, more preferred at least 90% (w/w).
  • Human SAM1a and SAM 1b are the clones NT2RP2000448 and NT2RM2001632, respectively.
  • a similar way of defining the MAS receptors or MAS signalling proteins of the present inven- tion is the similarity between the amino acid sequence of the various MAS receptors or MAS signalling proteins is at least about 90%, preferably about 95%, when compared with the amino acid sequence in SEQ ID NO: 2. Another expression of amino acid sequence similarity is homology. At the nucleotide level, the similarity between the nucleotides of the various MAS receptors or MAS signalling proteins is at least about 80%, preferably about 90%, when compared with the nucleotides in SEQ ID NO: 1.
  • high stringency conditions conditions under which the labeled probe, i.e., an oligonucleotide or polynucleotide of 25 or more contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3 will hybridize with high specificity to the polynucleotide sequences to be tested containing few, preferably less than 10%, more preferred less than 5%, if any, mismatched bases.
  • High-stringency hybridization conditions are described in, for example, Sambrook et al. 1989, “Molecular Cloning", Cold Spring Harbor Laboratory Press.
  • high stringency hybridization is achieved by incubating the probe and the membrane containing target DNA or mRNA in a buffer containing 6x SSC, 10% Dextran sulphate, 1% SDS, 5x Denhardts, 50 ⁇ g/ml salmon DNA (Stratagene), and 2 x 10 6 cpm/ml of the radiolabeled probe.
  • the incubation is at 68°C with shaking or rotation for at least 2 hours, typically overnight.
  • the mem- brane is then washed in 2x SSC, 0.1 % SDS at 42°C for 30 minutes, followed by a wash in 2x SSC, 0.1 % SDS at 68°C for 30 minutes, then a wash in 0.2 x SSC, 0.1 %SDS at 68°C, and finally a wash in 0.1 x SSC, 0.1% SDS at 68°C for 30 minutes.
  • the membrane is exposed to x-ray film.
  • oligonucleotide or polynucleotide probes 25-200 bases in length
  • high stringency hybridization is carried out in a solution containing 6x SSC, 0.05 M sodium phosphate (pH 6,8), 1 mM EDTA (pH 8,0), 5x Denhardts solution, 100 ⁇ g/ml salmon sperm DNA, 100 mg/ml dextran sulfate, and 180 pM of radiolabeled oligonucleotide (5x10 5 to 1.5x10 6 cpm/pmole).
  • the hybridization temperature vary depending on the length of the probe. Sambrook et al.
  • Hybridization is performed at 5-10°C less than the T m , and post-hybridization washes at 5°C below the T m , with the T m calculated as
  • Hybridization is done overnight with shaking or rotation.
  • the membrane is then washed twice with 2x SSPE, 0.1% SDS at room temperature for 15 minutes, then with 0.2x SSPE, 0.1% SDS 5°C below the T m of the probe, for 60 minutes.
  • the membrane is exposed to x-ray film.
  • test described in example 3 One way of determining the affinity constant of a MAS receptor or a MAS signalling protein is by the test described in example 3, below.
  • One way of determining whether FF-MAS binds to a specific MAS receptor is by the test described in example 4, below.
  • the test described in example 3 below can be used to determine whether a specific protein is a MAS receptor or a MAS signalling protein or, in other words, whether a specific protein is an analogue of SAM1a as specified in the claims below.
  • analogue is intended to indicate a naturally occurring variant (including one expressed in other animal species, for example, human, monkey, mouse or rat) of the MAS receptor or a "derivative", i.e., a polypeptide which is derived from the native MAS receptor by suitably modifying the DNA sequence coding for the variant, resulting in the addition of one or more amino acids at either or both the C- and N-terminal ends of the native amino acid sequence, substitution of one or more amino acids at one or more sites in the native amino acid sequence, deletion of one or more amino acids at either or both ends of the native sequence or at one or more sites within the native sequence, or insertion of one or more amino acids in the native sequence.
  • the invention provides means for regulating the MAS receptor/ligand interaction or MAS signalling protein/ligand interaction, and thus treating, therapeutically and/or prophylactically, a disorder which can be linked directly or indirectly to MAS receptor or MAS signalling protein or to its ligands, such as FF-MAS.
  • a disorder which can be linked directly or indirectly to MAS receptor or MAS signalling protein or to its ligands, such as FF-MAS.
  • agonists or antagonists may be identified which stimulate or inhibit, respectively, the interaction of ligand with MAS receptor or with MAS signalling protein.
  • the metabolism and reactivity of cells which express the receptor or signalling protein are controlled, thereby providing a means to control meiosis in order to treat infertility or to achieve a novel principle of contraception.
  • the invention provides screening procedures for identifying agonists or antagonists of events mediated by the ligand/MAS receptor or ligand/MAS signalling protein interaction.
  • Such screening assays may employ a wide variety of formats, depending to some extent on which aspect of the ligand, receptor or signalling protein interaction is targeted.
  • such assays may be designed to identify compounds which bind to the receptor or signalling protein and thereby block or inhibit interaction of the receptor or signalling protein and thereby block or inhibit interaction of the receptor or signalling protein with the ligand.
  • Other assays can be designed to identify compounds which can substitute for ligand and therefore stimulate MAS receptor-mediated or MAS signalling protein-mediated intracellular pathways.
  • Yet other assays can be used to identify compounds which inhibit or facilitate the association of MAS receptor or MAS signalling protein to FF-MAS and thereby mediate the cellular response to MAS receptor or MAS signalling protein ligand.
  • the initiation of fertilisation activation events are monitored in eggs which have been injected with, for example, mRNA which codes for MAS receptor or MAS signalling protein and subsequently exposed to selected compounds which are being screened, in conjunction with or apart form an appropriate ligand. See generally, Kline et al, Science 241 ( 988), 464-467, incorporated herein by reference.
  • the screening procedure can be used to identify reagents such as antibodies which specifically bind to the receptor or signalling protein and substantially affect its interaction with ligand, for example.
  • the antibodies may be monoclonal or polyclonal, in the form of antise- rum or monospecific antibodies, such as purified antiserum or monoclonal antibodies or mixtures thereof.
  • the antibodies are preferably substantially human to minimise immunogenicity and are in substantially pure form.
  • substantially human is meant generally containing at least about 70% human antibody sequence, preferably at least about 80% human, and most preferably at least about 90-95% or more of a human antibody sequence to minimise immunogenicity in humans.
  • Antibodies which bind to a MAS receptor or a MAS signalling protein may be produced by a variety of means.
  • the production of non-human antisera or monoclonal antibodies, for ex- ample, murine, lagomorpha equine, etc. is well known and may be accomplished by, for example, immunising the animal with the receptor or signalling protein molecule or a preparation containing a desired portion of the receptor or signalling protein molecule, such as that domain or domains which contributes to ligand binding.
  • monoclonal antibodies antibody-producing cells obtained from immunised animals are immortalised and screened, or screened first for the production of antibody which binds to the receptor or signalling protein and then immortalised.
  • DNA sequences which code for a human monoclonal antibody or portions thereof that specifically bind to the human re- ceptor or signalling protein by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., Science 246 (1989), 1275-1281 , incorporated herein by reference, and then cloning and amplifying the sequences which encode the antibody (or binding fragment) of the desired specificity.
  • the invention provides screening assays conducted in vitro with cells which express the receptor or signalling protein.
  • the DNA which encodes the receptor or signalling protein or selected portions thereof may be transfected into an established cell line, for example, a mammalian cell line such as BHK and CHO, using procedures known in the art (see, for example, Sambrook et al, Molecular Cloning, A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, which is incorporated herein by reference).
  • the receptor or signalling protein is then expressed by the cultured cells, and selected agents are screened for the desired effect on the cell, separately or in conjunction with an appropriate ligand such as FF-MAS or other MAS com- pounds.
  • Means for amplifying nucleic acid sequences which may be employed to amplify sequences encoding the receptor or signalling protein or portions thereof are described in US patent specification Nos. 4,683,195 and 4,683,202, incorporated herein by reference.
  • the screening assays provided by the invention relate to transgenic mammals whose germ cells and somatic cells contain a nucleotide sequence encoding MAS receptor protein or signalling protein or a selected portion of the receptor or signalling protein which, for example, binds ligand.
  • the screening assays provided by the invention relate to transgenic mammals where the nucleotide sequence encoding a MAS receptor or a MAS signalling protein is molecularly targeted to produce knock out animals with the phenotypical loss of the specific MAS signalling function.
  • the molecular knock out is tissue specific to gonadal tissue (ovary or testes) and is timely controlled in the development, thus inducible.
  • a sequence encoding, for example, the human MAS receptor may be introduced into a non-human mammalian embryo or, alternatively, knocked out, some of which are described in, for example, US pat- ent specification No. 4,736,866, Jaenisch, Science 240: 1468-1474 (1988) and Westphal et al, Annu. Rev. Cell Biol. 5: 181-196 (1989), which are incorporated herein by reference.
  • the animal's cells then express the receptor or signalling protein and thus may be used as a convenient model for testing or screening selected agonists or antagonists.
  • the invention concerns diagnostic methods and compositions.
  • MAS receptor or MAS signalling protein molecule By means of having the MAS receptor or MAS signalling protein molecule and antibodies thereto, a variety of diagnostic assays are provided. For example, with antibodies, including monoclonal antibodies, to MAS receptor or MAS signalling protein, the presence and/or concentration of receptor or signalling protein in selected cells or tissues in an individual or culture of interest may be determined. These assays can be used in the diagnosis and/or treatment of diseases such as, for example, male infertility, female infertility, or by means of contraception in both gender.
  • diseases such as, for example, male infertility, female infertility, or by means of contraception in both gender.
  • immunoassays Numerous types of immunoassays are available and are known to those skilled in the art, for example, competitive assays, sandwich assays, and the like, as generally described in, for example US Patent specification Nos. 4,642,285; 4,376,110; 4,016,043; 3,879,262; 3,852,157; 3,850,752; 3,839,153; 3,791 ,932; and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, N.Y. (1988), each incorporated by reference herein.
  • MAS receptor or MAS signalling protein is identified and/or quantified by using labelled antibodies, preferably monoclonal antibodies which are reacted with brain tissues, for example, ovarian or testicular tissue, oocyte preparations, or semen samples, and determining the specific binding thereto, the assay typically being performed under conditions conducive to immune complex formation.
  • Unlabeled primary antibody can be used in combination with labels that are reactive with primary antibody to detect the receptor or signalling protein.
  • the primary antibody may be detected indirectly by a labelled secondary antibody made to specifically detect the primary antibody.
  • the anti-MAS receptor-antibody or MAS signalling protein-antibody can be directly labelled.
  • labels such as radionuclides, particles (for example, gold, ferritin, magnetic particles, red blood cells), flourophores, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (particularly haptens).
  • RNA encoding the MAS receptor MAS signalling protein may be directly detected in cells with a labelled synthetic oligonucleotide probe targeting the MAS receptor RNA or MAS signalling protein RNA in a hybridisation procedure.
  • the polymerase chain reaction (Saiki et al, Science 239 (1988), 487, and US patent specification No. 4,683,195, each reference is hereby incorporated by reference) may be used to amplify DNA sequences, which are subsequently detected by their characteristic size on agarose gels, Southern blot of these gels using MAS receptor DNA or MAS signalling protein DNA or a oligonucleotide probe, or a dot blot using similar probes.
  • the probes may comprise from about 14 nucleotides to about 25 or more nucleotides, preferably, 40 to 60 nucleotides, and in some instances a substantial portion or even the entire cDNA of MAS receptor or MAS signalling protein may be used.
  • the probes are labelled with detectable signal, such as an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, and paramagnetic particle. High stringency in connection with hybridisation is obtained using the proper temperature and salt concentration.
  • Kits can also be supplied for use with the receptor or signalling protein of the subject inven- tion in the detection of the presence of the receptor or signalling protein or antibodies thereto, as might be desired in the case of autoimmune disease.
  • antibodies to MAS receptor or MAS signalling protein preferably monospecific antibodies such as monoclonal antibodies, or compositions of the receptor or signalling protein may be provided, usually in lyophilised form in a container, either segregated or in conjunction with additional reagents, such as anti-antibodies, labels, gene probes, polymerase chain reaction primers and polymerase, and the like.
  • the present invention relates to an isolated and/or purified polynucleotide molecule which hybridises at high stringency to an oligonucleotide or polynucleotide of 25 or more contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3 and which polynucleotide codes for a) a MAS receptor or for a MAS signalling protein; or b) a ligand binding domain of a MAS receptor or MAS signalling protein.
  • This polynucleotide may be a RNA antisense sequence or a cDNA sequence.
  • This polynucleotide may encode a polypeptide displaying MAS receptor or MAS signalling protein activity.
  • This polynucleotide may encodes a MAS receptor or MAS signalling protein (being able to bind to FF-MAS) having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the polynucleotide may have the nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3.
  • this invention relates to a probe of at least 12 nucleotides, said probe being capable of hybridising with nucleic acids which encode a MAS receptor or MAS signalling protein.
  • This probe may comprise an oligonucleotide or polynucleotide of 25 or more contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3 capable of specifically hybridising with a gene which encodes a MAS receptor or MAS signalling protein, or allelic and species variants thereof.
  • This probe may comprise from about 40 to about 60 nucleotides in length.
  • This probe may be labelled to provide a detectable signal.
  • This probe may comprise the nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3.
  • the present invention relates to a DNA construct comprising a DNA sequence which hybridises at high stringency to an oligonucleotide or polynucleotide of 25 or more contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3 and which encodes a) a MAS receptor or MAS signalling protein; or b) a ligand binding domain of a MAS receptor or MAS signalling protein.
  • This DNA construct may have a DNA sequence encoding a MAS receptor or MAS signalling protein having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the present invention relates to a cultured cell line, yeast or bacteria transformed or transfected with a DNA construct which comprises a DNA sequence which hybridises at high stringency to an oligonucleotide or polynucleotide of 25 or more contiguous nu- cleotides of SEQ ID NO: 1 or SEQ ID NO: 3 and which encodes a) a MAS receptor or MAS signalling protein; or b) a ligand binding domain or a transmembrane domain of a MAS receptor or MAS signalling protein.
  • This cell line, yeast or bacteria may not express endogenous MAS receptor or MAS signalling proteins.
  • the MAS receptor or MAS signalling protein, a peptide fragment thereof or a salt thereof according to the present invention may be isolated and/or purified.
  • the isolated and/or purified protein (MAS receptor or MAS signalling protein) may comprise the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the present invention relates to an isolated antibody which specifically binds to a MAS receptor or MAS signalling protein.
  • said antibody may be a monoclonal antibody.
  • This isolated antibody may block the binding of MAS to a MAS receptor or MAS signalling protein.
  • the present invention relates to a hybridoma which produces a monoclonal antibody as mentioned herein.
  • the present invention relates to a method for detecting the presence of a compound or a salt thereof which has affinity for a MAS receptor or MAS signalling protein, com- prising the steps of a) contacting the compound with the MAS receptor or MAS signalling protein, a peptide fragment thereof or a salt thereof; and b) measuring the affinity of said compound for the MAS receptor or MAS signalling protein.
  • This method for detecting the presence of MAS antagonists may comprise the steps of a) exposing a compound in the presence of a MAS agonist including MAS (FF-MAS) to a MAS receptor or MAS signalling protein coupled to a response pathway under conditions and for a time sufficient to allow binding of the compound to the MAS receptor or MAS signalling protein and an associated response through the pathway; and b) detecting a reduction in the stimulation of the response pathway resulting from the binding of the compound to the MAS receptor or MAS signalling protein , relative to the stimulation of the response pathway by the MAS agonist alone and there from determining the presence of a MAS antagonist.
  • a MAS agonist including MAS FF-MAS
  • a method for detecting the presence of MAS agonists may comprise the steps of a) exposing a compound in the presence of a MAS antagonist to a MAS receptor or MAS signalling protein coupled to a response pathway under conditions and for a time sufficient to allow binding of the compound to the MAS receptor or MAS signalling protein and an associated response through the pathway; and b) detecting an increase of the stimulation of the response pathway resulting from the binding of the compound to the MAS receptor or MAS signalling protein, relative to the stimulation of the response pathway by the MAS antagonist alone and there from determining the presence of a MAS agonist.
  • the present invention relates to a compound or a salt thereof which has affinity for the MAS receptor or a MAS signalling peptide and which compound or salt is detected by a method described herein.
  • the present invention relates to a method for producing a MAS receptor or MAS signalling protein having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, which may comprise a) growing cells, yeast or bacteria transformed or transfected with a DNA construct which comprises a DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 coding for the expression of the MAS receptor or MAS signalling protein, and b) isolating the MAS receptor or MAS signalling protein from the cells.
  • the MAS receptor or MAS signalling protein may be isolated by immunoaffinity purification.
  • the present invention relates to a kit for screening a compound or a salt thereof which has affinity for a MAS receptor or MAS signalling protein, which contains the MAS receptor or MAS signalling protein, the peptide fragment thereof or a salt thereof.
  • the MAS receptor or MAS signalling protein may be defined as one which comprises the amino acid sequence shown in SEQ ID NO: 2, or an analogue thereof binding FF- MAS, with an affinity constant below 100 ⁇ M, preferably below 10 ⁇ M.
  • the MAS receptor or MAS signalling protein comprising the amino acid sequence shown in SEQ ID NO: 2, or an analogue thereof binding FF-MAS, with an affinity constant below 100 ⁇ M, preferably below 10 ⁇ M may be different from the amino acid sequence in SEQ ID NO: 6 and 8.
  • the MAS receptor or MAS signalling protein may comprise the amino acid sequence shown in SEQ ID NO: 4, or an analogue thereof binding FF-MAS, with an affinity constant below 100 ⁇ M, preferably below 10 ⁇ M.
  • the MAS receptor or MAS signalling protein comprising the amino acid sequence shown in SEQ ID NO: 4, or an analogue thereof binding FF-MAS, with an affinity constant below 100 ⁇ M, preferably below 10 ⁇ M, may be different from the amino acid sequence in SEQ ID NO: 6 and 8.
  • the MAS receptor or MAS signalling protein may comprise the partial amino acid sequence shown in SEQ ID NO: 6, or an analogue thereof binding FF-MAS, with an affinity constant below 100 ⁇ M, preferably below 10 ⁇ M.
  • the MAS receptor or MAS signalling protein comprising the partial amino acid sequence shown in SEQ ID NO: 6, or an analogue thereof binding FF-MAS, with an affinity constant below 100 ⁇ M, preferably below 10 ⁇ M, may be different from the amino acid sequence in SEQ ID NO: 6 and 8.
  • the MAS receptor or MAS signalling protein may comprise the partial amino acid sequence shown in SEQ ID NO: 8, or an analogue thereof binding FF-MAS, with an affinity constant below 100 ⁇ M, preferably below 10 ⁇ M.
  • the MAS receptor or MAS signal- ling protein comprising the partial amino acid sequence shown in SEQ ID NO: 8, or an analogue thereof binding FF-MAS, with an affinity constant below 100 ⁇ M, preferably below 10 ⁇ M, may be different from the amino acid sequence in SEQ ID NO: 6 and 8.
  • the MAS receptor or MAS signalling protein according to the present invention may be a soluble and purified protein which is present in a buffer suitable for detecting ligands, for example by a binding assay.
  • the MAS receptor or MAS signalling protein being a soluble and purified protein which is present in a buffer suitable for detecting ligands, for example by a binding assay, may be different from the amino acid sequence in SEQ ID NO: 6 and 8.
  • the present invention relates to a DNA construct which comprises a DNA sequence encoding a MAS receptor or MAS signalling protein as described herein or a DNA sequence coding for a functional analog thereof binding to FF-MAS.
  • the DNA construct comprises a DNA sequence encoding a MAS receptor or MAS signalling protein as defined herein or a DNA sequence coding for a functional analog thereof binding to FF-MAS may be different from the nucleotides of SEQ ID NO: 5 and 7.
  • the DNA construct of the present invention may comprise the DNA sequence shown in SEQ ID NO: 1 or a fragment thereof, or a DNA sequence coding for a functional analogue thereof binding FF-MAS, with an affinity constant below 100 ⁇ M, preferably below 10 ⁇ M.
  • This DNA construct comprising the DNA sequence shown in SEQ ID NO: 1 or a fragment thereof, or a DNA sequence coding for a functional analogue thereof binding FF-MAS, with an affinity constant below 100 ⁇ M, preferably below 10 ⁇ M, may be different from the nucleotides of SEQ ID NO: 5 and 7.
  • the DNA construct according to the present invention may comprise the partial DNA sequence shown in SEQ ID NO: 5, or a DNA sequence coding for a functional analogue thereof binding FF-MAS, with an affinity con- stant below 100 ⁇ M, preferably below 10 ⁇ M.
  • This DNA construct comprising the partial DNA sequence shown in SEQ ID NO: 5, or a DNA sequence coding for a functional analogue thereof binding FF-MAS, with an affinity constant below 100 ⁇ M, preferably below 10 ⁇ M, may be different from the nucleotides of SEQ ID NO: 5 and 7.
  • the present invention relates to a recombinant expression vector which carries an inserted DNA construct according to any one of the preceding claims to a DNA construct.
  • the present invention relates to a cell containing a recombinant expression vector as defined herein.
  • This cell may contain a DNA construct as defined herein integrated in its ge- nome.
  • This cell may be a eukaryotic cell, in particular an insect or a mammalian cell.
  • the present invention relates to a method of screening for ligands to the MAS receptor, i.e., agonists or antagonists of FF-MAS activity, the method comprising incubating a MAS receptor or MAS signalling protein as defined herein with a substance suspected to be an agonist or antagonist of FF-MAS, and subsequently with FF-MAS, or an analogue thereof, and detecting any effect of binding of FF-MAS, or the analogue to the MAS receptor or MAS signalling protein.
  • the method of screening for ligands to the MAS receptor may comprise incubating FF-MAS, or an analogue thereof with a substance suspected to be an agonist or antagonist of activity of FF-MAS, and subsequently with a MAS receptor or MAS signalling protein as described herein, and detecting any effect of binding of FF-MAS, or the analogue to the receptor.
  • the present invention relates to the use of a MAS receptor or MAS signalling pro- tein as defined herein for screening for agonists or antagonists of activity of FF-MAS.
  • the present invention relates to the use of DNA constructs as defined herein for isolation of tissue and/or organ specific variants of the MAS receptor or MAS signalling protein.
  • the present invention relates to the use of a MAS receptor or MAS signalling protein isolated as described herein.
  • Two antisense oligonucleotides (20 nucleotides) were utilized for microinjection: 5'-TCCAC- GATGGACGCCATCTT-3' and 5'-GCCAGCAGGAGAGCCATTCG-3', complementary to the kozak sequence of the mRNA encoded by the cDNA sequence herein designated SAM1a and SAMlb, respectively, both of which are defined in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, shown below.
  • the corresponding sense oligonucleotides were microinjected: 5'-AAGATGGCGTCCATCGTGGA-3' and 5'-CGAATGGCTCTC- CTGCTGGC-3' for mRNA SAM1a and SAMlb, respectively.
  • SAM1a antisense was co- injected with SAMlb antisense from a stock solution containing 1.25 ⁇ g/ ⁇ l of each nucleotide in 10 % human serum albumin (hereinafter designated HSA) plus 5 mM Tris (pH value: 7.5).
  • HSA human serum albumin
  • SAM 1a sense was co-injected with SAMlb sense from a stock solution containing 1.25 ⁇ g/ ⁇ l of each nucleotide in 10 % HSA plus 5 mM Tris (pH value: 7.5).
  • each oligonucleotide (10 pi) were injected into the cytoplasma of individual germinal vesicle (GV)-stage oocytes loaded in a droplet of alpha-MEM supplemented with 0.8% HSA and 3 mM hypoxantine under mineral oil in a 35 mm petri dish on the stage of an inverted micro- scope.
  • the oocytes were obtained from the ovaries of 21-24 days old mice following 48 hours priming with follicle stimulating hormone (hereinafter designated FSH) as described by Gr ⁇ ndahl et al. 1998 in Biol. Reprod. 58 (1998), 1297 et seq.
  • FSH follicle stimulating hormone
  • Oocytes were sucked on to a holding pipette (120 ⁇ M outer diameter and 20 ⁇ m inner diameter) and an injection pipette (Eppendorph, Hamburg, Germany) was fitted to a pressure microinjector (Eppendorph, Hamburg, Germany).
  • the pipette holder was attached to a piezoelectric positioning system (Burleigh, NY, USA) mounted on a motorized micromanipulator (Luigs and Neumann, Ratin- gen, Germany).
  • the injection pipette was pushed against the zona pelludica, and then a piezoelectric pulse was given, moving the injection pipette 20 ⁇ m forward.
  • GVBD germinal vesicle breakdown
  • GVBD was inhibited by 50% in antisense injected oocytes compared to control (i.e., sense injected and non-injected oocytes).
  • This result indicates a selective inhibition of the mRNAs coding for SAM1a and SAM1 b by the antisense probe.
  • SAM1a and SAMlb proteins are crucial involved in the MAS sig- nailing, since a functional knock out of ofe novo protein synthesis of these molecules partly disrupt the MAS signals in oocytes.
  • SAM1a and SAMlb are two closely related proteins originating from the same gene which possesses complementary functions regarding MAS signalling in oocytes.
  • Assay to determine whether a specific polynucleotide encodes a protein which is a MAS receptor or a MAS signalling protein 10 ⁇ l of the unlabelled FF-MAS (1 , 3, 10, 30, 100, 300, 1000, 3000 nM in 6.6% EtOH) is mixed with 10 ⁇ l of 200 nM 3 H-labelled FF-MAS (approximately 12.8 Ci/mmol) in assay buffer (10 mM Tris; 1.5 mM EDTA; 10% glycerin; 1.0 mM CHAPS; 1% BSA). 10 ⁇ g of the specific protein to be tested freshly diluted in assay buffer, is added to give a final volume of 40 ⁇ l.
  • Unspecific binding is measured in the presence of 30 ⁇ M unlabelled FF-MAS, total binding is determined by adding 10 ⁇ l assay buffer containing 6.6 % EtOH. Incubation is performed for 2 hours at 4°C. 250 ⁇ l of ice-cold assay buffer containing 2 % Cab-osil M-5 and 0.2 % dextran is added to each tube, mixed and spinned briefly. Approximately 5 minutes after adding Cab-osil M-5, the tubes are centrifuged for 5 minutes, 14000 rpm (minifuge). 200 ⁇ l of the supernatant is transferred to a microscint-tube and 3.5 ml atomlight is added. Tubes are measured in a liquid scintillation counter. If 3 H FF-MAS binding can be displaced by unlabelled FF-MAS, then the protein tested is a MAS receptor or a MAS signalling protein.
  • 10 ⁇ l of the compound to be tested (1 , 3, 10, 30, 100, 300, 1000, 3000 nM in 6.6% EtOH) is mixed with 10 ⁇ l of 200 nM 3 H-labelled FF-MAS (approximately 12.8 Ci/mmol) in assay buffer (10 mM Tris; 1.5 mM EDTA; 10% glycerin; 1.0 mM CHAPS; 1% BSA).
  • 10 ⁇ g of SAM1a protein freshly diluted in assay buffer is added to give a final volume of 40 ⁇ l. Unspecific binding is measured in the presence of 30 ⁇ M unlabelled FF-MAS, total binding is determined by adding 10 ⁇ l assay buffer containing 6.6% EtOH. Incubation is performed for 2 hours at 4°C.
  • cDNA library was prepared from mRNA isolated from 10,000 oocytes, from 24 days old mice.
  • the cDNA library was constructed in the pSPORT plasmid vector (LifeTechnologies). Clones were picked at random and partially sequenced, and the sequences were assembled using phred/phrap programs. Out of several thousand clones that were sequenced, an assembly of two exhibited 21 % amino acids identity to a human Oxysterol Binding Protein. The longest clone MOCY2864 was completely sequenced and no identical or orthologous genes were found in the databases. This new gene was named SAM1.
  • Amplification of the 5' end of SAM1 cDNA was performed by PCR on the oocyte library using a primer specific for pSPORT, #176959 (SEQ ID NO: 9) and a primer specific for SAM1 #198241 (SEQ ID NO: 10). This revealed cDNAs with two different 5' ends, which were designated SAM1a and SAMlb.
  • Full-length PCR amplification was done on the mouse oocyte library using primer #199772 (SEQ ID NO: 11) and #198239 (SEQ ID NO: 12) for SAM1a and #201790 (SEQ ID NO: 13) and #198239 (SEQ ID NO: 12) for SAMlb.
  • SAM1a and SAMb cDNAs were digested with Nhel and Notl restriction enzymes and cloned into pcDNA3,1+ (Invitrogen).
  • SAM1a and SAMlb cDNAs were PCR amplified using the primers #199772 (SEQ ID NO: 11) and #211465 (SEQ ID NO: 14) and primers #201790 (SEQ ID NO: 13) and #211465 (SEQ ID NO: 14), respectively. Recognition sites for Nhel and Xmal, respectively, were incorporated in the primers.
  • PCR-products were then cloned into pBlueBac4,5V5HIS (Invitrogen) using the restriction enzymes Nhel and Xmal.
  • This intermediate construct was then digested by Smal and BstBI, filled-in using the Klenow fragment of DNA polymerasel and then religated.
  • SAM1a in pBlueBac4,5V5HIS and "SAMlb in pBlueBac4,5V5HIS”.
  • SAM1a-HIS and SAM1b-HIS proteins were expressed using recombinant Baculo virus in Sf9 cells according to the "Bac-N-BlueTM Transfection Kit” manual (Invitrogen).
  • Bac-N-BlueTM DNA and the recombinant transfer plasmid "SAM1 in pBlueBac4,5V5HIS" (4 ⁇ g) were incubated with 1 ml Grace's Insect Media and and 20 ⁇ l Insectin-Plus Liposomes for 15 minutes, then the mixture was added to 2x10 6 Sf9 cells in a 60 mm dish. The cells were left for 96 hours rocking at 27°C.
  • Vira were isolated and plaque assay was performed. Putative recombinant plaques were picked and P-1 viral stocks were made. PCR analysis of recombinant viral clones was done and from positive clones high-titer viral stocks were then prepared.
  • 500 ml Sf9 cells (2,0 x 10 6 cells/ml) was infected with 25 ml virus (1 ,8 x 108 plaque forming units/ml) or 60 ml virus (6 x 10 7 pfu/ml) for SAMIa and SAMl b, respectively. After 70 hours of incubation the cells were pelleted by centrifugation and the protein was purified.
  • HIS-SAM1a and HIS-SAM1b Purification of HIS-SAM1a and HIS-SAM1b was performed according to the manual of QIAGEN: The QIAexpressionist forth edition.
  • cell cultures of SF9 insect cells (containing the construct for 6xHis-SAM1a or 6xHis- SAM1b in a baculovirus expression vector) were centrifuged, pellets were lysed by addition of lysis buffer (50 mM NaH 2 P0 4 , 300 mM NaCI, 10 mM imidazole, pH 8) and lysozyme, sonication on ice 6 x 10 seconds, where after the lysates were cleared by centrifugation.
  • lysis buffer 50 mM NaH 2 P0 4 , 300 mM NaCI, 10 mM imidazole, pH 8

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Abstract

Cette invention a trait à l'identification de deux stérols stimulant la méiose, dénommés SAM1a et SAM1b.
EP01957793A 2000-08-25 2001-08-24 Deux recepteurs d'un sterol activateur de meiose designes sam1a et sam1b Withdrawn EP1315751A2 (fr)

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DK200001259 2000-08-25
DKPA200001259 2000-08-25
WOPCT/DK01/00550 2001-08-20
PCT/DK2001/000550 WO2002016432A2 (fr) 2000-08-25 2001-08-20 Sam
PCT/DK2001/000558 WO2002016433A2 (fr) 2000-08-25 2001-08-24 Sam

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AU2003203145A1 (en) * 2002-02-22 2003-09-09 Novo Nordisk A/S A transducer of mas signalling
TW201221505A (en) 2010-07-05 2012-06-01 Sanofi Sa Aryloxyalkylene-substituted hydroxyphenylhexynoic acids, process for preparation thereof and use thereof as a medicament

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