US20050100972A1 - Use of grf1 protein for screening molecules - Google Patents

Use of grf1 protein for screening molecules Download PDF

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US20050100972A1
US20050100972A1 US10/344,223 US34422303A US2005100972A1 US 20050100972 A1 US20050100972 A1 US 20050100972A1 US 34422303 A US34422303 A US 34422303A US 2005100972 A1 US2005100972 A1 US 2005100972A1
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grf1
protein
cells
ras
yeast
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Jean-Michel Itier
Marie-Christine Multon
Gwenaelle Ret
Jean-Marie Stutzmann
Florence Wahl
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Aventis Pharma SA
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Aventis Pharma SA
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Assigned to AVENTIS PHARMA S.A. reassignment AVENTIS PHARMA S.A. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ITIER, JEAN-MICHEL, RET, GWENAELLE, MULTON, MARIE-CHRISTINE, STUTZMANN, JEAN-MARIE, WAIIL, FLORENCE
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to the field of biology and of cell signaling in neurons. More specifically, the present invention relates to novel uses of all or part of GRF1 (Guanine Nucleotide Releasing Factor 1) protein for screening molecules exhibiting an activity of protection against neuronal death and of treatment for obesity.
  • GRF1 protein which is represented diagrammatically in FIG. 1A , was originally discovered in humans for its ability to modulate the activation state of p21Ras protein.
  • the sequences of the human, mouse and rat GRF1 proteins are, respectively, the sequences SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.
  • Application WO 93/21314 and U.S. Pat. No. 5,656,595 describe the identification, isolation and also characterization of the human form of this protein.
  • GRF1 has several functional domains, termed PH, IQ, DH and CDC25 domains, the meaning and involvement of which are specified hereinafter.
  • GRF1 comprises a DH (Db1 homology) domain which exhibits strong sequence homology with a region of the proto-oncogene Db1.
  • Db1 is an exchange factor for the small G proteins of the Rho/Rac/Cdc42Hs family involved in the regulation of various cell signals controlling either cytoskeletal motility or the activation of “stress” kinases (1).
  • the activity of exchange of GDP/GTP on the proteins of the Rho family is carried by the DH domain.
  • This domain is found in other low molecular weight G protein exchange factors, including the GRF1 proteins and the SOS proteins.
  • Results have been published which suggest that the DH domain of GRF1 is functional on Rac (activation of Rac in GTP form) and leads to activation of JNK1 in cells in culture which overexpress the beta/gamma subunit of heterotrimeric G proteins (2).
  • the functional role of the DH domain of GRF1 in neuronal processes has also been suggested by the results of site-directed mutagenesis experiments which show that activation of GRF1 protein by calcium requires an intact DH domain (3, 4).
  • GRF1 As regards the exchange activity of GRF1 for the Ras proteins (Ha, Ki and N-Ras), it is carried by the region homologous to CDC25 of Saccharomyces cerevisiae , located at the carboxy-terminal end of the molecule (6).
  • GRF1 has two PH (Pleckstrin Homology) domains which are domains of interaction between proteins and which are probably involved in binding to the beta-gamma subunits of heterotrimeric G proteins (7-9).
  • GRF1 protein The functions of the GRF1 protein, initially discovered for its exchange activity on the proto-oncogene Ras, are today known in greater detail. Contrary to the SOS1, SOS2 and GRF2 proteins, three other exchange factors for Ras which are expressed ubiquitously, it is now accepted that expression of the grf1 gene is restricted to the central nervous system in humans, mice and rats (6, 10, 11).
  • mice In the brain, the regionalization of this expression has been finely studied. In the rat, it is considerable in the hippocampus, the neocortex, some deep nuclei and the granule cells of the anterior lobe of the cerebellum (12). In mice, it has also been described as being abundant in the amygdala (13) and the hypothalamus (14).
  • the grf1 gene appears to be restricted to neurons (15). This gene undergoes strict transcriptional control over time, since it is not expressed during embryonic life and only begins to be transcribed at the time of birth, to reach a maximum level around the fifteenth day (14, 15).
  • grf1 The expression of grf1 is controlled by a particular transcriptional mechanism called parental imprinting, which, in mice, results in expression of the gene from the allele of paternal origin, whereas the maternal allele is silent (14, 16). It is interesting to note that some genes (proto-oncogenes) normally subjected to parental imprinting are involved in cell proliferation and the appearance of tumors when there is a loss of the imprinting mechanism and the two alleles are transcribed.
  • GRF1 does not transduce the signals resulting from the binding of ligands to receptors with tyrosine kinase activity, but rather those derived from heterotrimeric G protein-coupled receptors with seven transmembrane domains (17-19).
  • the applicant has shown, by studying mice carrying an inactivating mutation of the grf1 gene, that the absence of expression of the GRF1 protein imparts protection against physical damage to the brain during cerebral ischemia.
  • Leptin is a cytokine associated with the feeling of satiety which plays a major role in controlling weight gain (20).
  • the applicant set out to search for compounds intended for the prevention and/or treatment of disorders affecting in particular the central nervous system and obesity.
  • the present invention therefore relates to the use of all or part of the GRF1 protein, or of cells expressing all or part of the GRF1 protein, in methods for detecting compounds intended for the prevention and/or treatment of pathological conditions or of disorders of the central nervous system involving neuronal death, such as apoptosis, or associated with leptin metabolism. It also relates to compounds intended for the prevention and/or treatment of pathological conditions or of disorders of the central nervous system involving neuronal death, or associated with obesity or with leptin metabolism.
  • the GRF1 protein is preferentially of human origin. It may, however, be of any other origin, and in particular may be mouse GRF1 protein or the GRF1 protein for any other mammal. It may also be any protein exhibiting at least 85%, and preferentially 90%, identity with a GRF1 protein, and in particular with the human GRF1 protein having the sequence SEQ ID No. 1, or with a GRF1 protein of animal origin, such as those having the sequences SEQ ID No. 2 or SEQ ID No. 3.
  • the expression “part of the GRF1 protein” is intended to mean an amino acid sequence comprising a functional part of the GRF1 protein, and in particular the sequences corresponding to all or part of one of the PH, DH or CDC25 domains of the GRF1 proteins.
  • One of the advantages of the screening methods which are the subject of the present invention lies in particular in the position of GRF1 upstream of the JNKs ( FIG. 1B ), which has the advantage that it is possible to specifically regulate the JNK activation pathways which would involve GRF1.
  • Another advantage lies in the demonstration of the lack of toxicity of these compounds, the grf1-knockout mice being viable and in good health.
  • the screening method according to the present invention using all or part of the GRF1 protein may comprise steps consisting of measuring:
  • the present invention relates to a method for screening or for detecting compounds intended for the prevention and/or treatment of pathological conditions of the central nervous system involving neuronal death or associated with leptin metabolism, comprising the steps consisting in:
  • the cell lyzate obtained in step (ii) can be incubated in microtitration plate wells precoated with an anti-GRF1 antibody.
  • the GRF1 phosphorylation can also be measured using an antibody specific for phosphorylated amino acid.
  • the present invention relates to a method for screening or for detecting compounds intended for the prevention and/or treatment of pathological conditions of the central nervous system involving neuronal death or associated with leptin metabolism, comprising the steps consisting in:
  • the amount of phosphorylated GRF1 is preferentially measured using an antibody specific for a phosphorylated amino acid.
  • Said cells can be cultured in a medium containing orthophosphate labeled with P 32 or with P 33 .
  • the phosphorylation of the GRF1 protein can be carried out in said neuronal cells by adding carbachol to the culture medium, or else in cell lines starved of serum overnight and then re-incubated in the presence of serum, or alternatively in cell lines cotransfected with cDNAs encoding the trimeric G protein beta-gamma subunits ⁇ 1 ⁇ 2 or ⁇ 1 ⁇ 5, then starved of serum overnight and then reincubated in the presence of serum.
  • the screening comprises measuring the exchange activity of GRF1 on certain proteins of the small G protein family (Ras, Rac, Cdc42).
  • the present invention relates to a method for screening or for detecting compounds intended for the prevention and/or treatment of pathological conditions of the central nervous system involving neuronal death or associated with leptin metabolism, comprising the steps consisting in:
  • Such a protein of the small G protein family may in particular be Ras (53, 54, 55), Rac (56, 57, 58) or Cdc42 (59).
  • the exchange activity of the GRF1 protein on proteins of the Ras family, or the Rac and CDC42 proteins can be measured in vitro in an acellular medium by incubating, in microtitration plate wells, a reaction mixture comprising said small G proteins in the recombinant state, loaded with tritiated GDP, cold GTP, and all or part of the GRF1 protein in the recombinant state. An aliquot fraction of said reaction mixture is taken and filtered over a membrane and the radioactivity which is retained thereon is measured, or else the content of each of said wells is passed over a PD10 column and the radioactivity of the eluate is measured.
  • Said small G proteins in the recombinant state may be chosen from the group consisting of the wild-type Ras, Cdc42 or Rac proteins either in the form of fusion proteins and expressed in E. coli or in mammalian cells, or in a tagged form in a baculovirus, expressed in insect cells and purified.
  • the amount of nucleotides exchanged can also be demonstrated by retention.
  • GST-Raf (RBD domain) or GST-PAK (CRIB domain) fusion proteins, and a mixture of anti-GST IgG and SPA (scintillation proximity assay) protein A PVT coupled to microspheres (Amersham), are added and the fluorescence of said microtitration plates is measured after centrifugation.
  • the GDP-bound forms do not allow interaction.
  • the sequences of PAK and Raf have been published (see respectively references 60 to 64, and 65 to 69).
  • the Ras (or Rac) recombinant protein is incubated with cold GDP. Ras-GDP (or Rac-GDP) forms. Tritiated GTP and GRF1 are then added. The exchange reaction takes place. Ras-tritiated GTP forms. After 60 minutes, a GST-Raf fusion protein is added (or GST-PAK if working with Rac). The fusion protein interacts with Ras-GTP, the amount of which in the medium is dependent on the exchange activity of GRF1.
  • the microspheres which are coupled to an anti-GST antibody, are added. The GST-Raf/Ras-tritiated GTP complex attaches to the spheres. These spheres have the ability to scintillate when they are in the proximity of a radioactive source. They then make it possible to quantify the proportion of Ras-GTP and therefore the exchange activity of GRF1 in the medium.
  • the screening method can also be used in a cellular system.
  • the membrane of such a yeast is permeabilized beforehand and/or at least one of the genes involved in the mechanisms of detoxification has been inactivated.
  • the screening method may also be a “double-hybrid” system of protein-protein interaction comprising a first hybrid protein consisting of a protein from fusion between Ras, CDC42Hs or Rac and a DNA-interacting domain, and a second hybrid protein consisting of a protein from fusion between all or part of GRF1 and a transactivating domain. It may also be a “double-hybrid plus one” system consisting in expressing, in the nucleus of the same S.
  • GRF1 GRF1
  • PAK1 CRIB domain
  • CDC42Hs CRIB domain
  • RBD c-Raf1
  • the screening may be a cellular screening comprising the steps consisting in
  • the present invention relates to the use of compounds, as screened by one of the detection methods mentioned above, in the preparation of medicinal products.
  • the present invention relates to the use of mice carrying an inactivating mutation of the grf1 gene, as a model for studying the prevention of pathological conditions or of disorders involving neuronal death or associated with leptin metabolism.
  • the subject of the present application is to search for compounds intended for the production of medicinal products for the prevention and/or treatment of various pathological conditions of the central nervous system (A) and of obesity (B). Because of the central role of GRF1, the methods which are the subject of the present application can also be used to screen molecules exhibiting activity against certain cardiovascular diseases (B), and pathological angiogenic processes (C), and with respect to other biological molecules (D).
  • the present invention makes it possible to screen compounds which antagonize the effect of the activity of GRF1 and which impart protection against neuronal death induced by cerebral ischemia, cranial or cerebral trauma, and spinal or medullary trauma.
  • the methods which are the subject of the present invention can be used to screen molecules for the treatment or prevention of neurodegeneration involving neuronal apoptosis, of Parkinson's disease, of Alzheimer's disease, of senile dementia, of Huntington's chorea, of amyotrophic lateral sclerosis, of epilepsy, of multiple sclerosis, of cerebella and spinal cerebella disorders, of cognitive disorders, of cranial trauma, of medullary trauma, of traumas of the inner ear, of retinal traumas, of glaucomas, and of cancers of the nervous system.
  • neurodegeneration involving neuronal apoptosis of Parkinson's disease, of Alzheimer's disease, of senile dementia, of Huntington's chorea, of amyotrophic lateral sclerosis, of epilepsy, of multiple sclerosis, of cerebella and spinal cerebella disorders, of cognitive disorders, of cranial trauma, of medullary trauma, of traumas of the inner ear, of retinal trauma
  • the methods which are the subject of the present invention can be used to screen molecules for the treatment or prevention of psychoses including schizophrenia, of anxious disorders, of depression, of panic attacks, of peripheral neuropathies, of migraine, of shaking, of obsessive-compulsive disorder, of thymic disorders, of tardive dyskinesia, of bipolar disorders, of drug-induced movement disorders, of dystonias, of endotoxemic shocks, of hemorrhagic shocks, of hypotension, of insomnia, of immunological diseases, of vomiting, of appetite disorders (bulimia, anorexia), of obesity, of memory disorders, in withdrawal from chronic treatment and alcohol abuse or drug abuse (opioids, barbiturates, cannabis, cocaine, amphetamine, phencyclidine, hallucinogens, benzodiazepines for example), as analgesics or potentiators of the analgesic activity of narcotic and non-narcotic medicinal products.
  • psychoses including schizophrenia, of anxious disorders, of depression, of panic attacks, of peripheral neuro
  • the present invention makes it possible to search for compounds which antagonize the activity of GRF1 and which play a protective role against weight gain, mainly in adult individuals.
  • GRF1 is thought to be involved in the upstream regulation of leptin synthesis, directly or indirectly.
  • Leptin is a hormone known to inhibit the release of Neuropeptide-Y (NPY), an appetite-stimulating molecule produced by the neurons of the arcuate nucleus of the hypothalamus (20, 21). It also controls the synthesis of an anorectic peptide: CART (cocaine- and amphetamine-regulated transcript) in the arcuate nucleus (22). The antagonist activities of these two neuropeptides consequently balance the effect of the leptin signal on food intake. Leptin also stimulates the alpha-MSH/melanocortin 4-receptor anorectic circuit (23-25).
  • GRF1 is liable to contribute to the signal in pathways which use the neuromediators (which bind to receptors with seven transmembrane domains).
  • the absence thereof in our mutant mice might result in a signal of the “satiety and/or increase in the expending of energy” type with, consequently, a decrease in fat mass and a low leptinemia.
  • the vascular endothelium appears to be a new target for leptin. Recent results show that leptin stimulates endothelial cell proliferation and angiogenesis in vivo (26, 27).
  • Angiogenesis plays an important role in embryogenesis, cicatrization and the menstrual cycle, but also in pathological situations such as tumor vascularization, rheumatoid arthritis, psoriasis, Kaposi's sarcoma, diabetic retinopathies and atherosclerosis.
  • the present inventors have shown that adult mice knockout for the grf1 gene have a leptin content much lower than that of control animals. It is possible that the mutant animals exhibit a certain form of protection against the appearance of pathological angiogenic processes and, consequently, against the growth and dissemination of tumors and against all the abovementioned pathological conditions.
  • GRF1 antagonists which would lead to a decrease in leptinemia might have a protective role against these diseases.
  • a contraceptive role can also be envisioned.
  • Leptin is involved in triggering puberty and controlling reproduction (28-32).
  • Leptin also plays a role in regulating the T-lymphocyte-regulated immune response (33).
  • Modulators of GRF1 activity might have an effect on the immune function and on mechanisms of reproduction.
  • the latter hypothesis appears, moreover, to be verified, since the observations of the present inventors indicate delayed puberty and early menopause in the grf1 KO mouse.
  • This phenomenon may be due in part to the decrease in leptin level or to deregulation of synthesis of sex hormones controlled by the hypothalamo-hypophyseal axis.
  • the inhibitory properties of the leptin with respect to bone synthesis have recently been demonstrated. It acts by inhibiting the activity of osteoblasts, a population of cells responsible for bone formation (34). Modifying leptinemia by acting on GRF1 might make it possible to treat diseases associated with a decrease in bone density, such as, for example, osteoporosis, or conversely those associated with considerable calcification, such as for example osteopetrosis.
  • mice knockout for the NPY gene exhibit a pronounced taste for alcohol and are more resistant to its sedative and hypnotic effects than wild-type mice (35).
  • Leptin plays a negative role on NPY release (20, 21).
  • Modulators of GRF1 activity might be used in the treatment of alcoholic behavior and in the treatment of sleep disorders.
  • FIG. 1A is a diagrammatic representation of GRF1 protein
  • FIG. 1B is a diagram combining the main signaling pathways which involve GRF1 protein function
  • FIGS. 2A and 2B are histograms representing neurological examinations
  • FIGS. 3A and 3B are histograms illustrating the extent of the brain lesions respectively overall and of the cortex
  • FIG. 3C illustrates the lesions of the various areas of the cortical region
  • FIG. 3D illustrates the volume of the lesions of the cortical region between 3.22 and 1.76.
  • FIGS. 4A and 4B illustrate, respectively, the weight and the leptinemia of grf1 KO mutant mice (noted ⁇ / ⁇ ) and wild-type mice (noted +/+), and
  • FIGS. 5A and 5B illustrate, respectively, the weight and the leptinemia of grf1 KO mutant mice and wild-type mice after fasting for 48 h.
  • the grf1 KO mice offer the possibility of investigating the impact of the GRF1 protein-dependent pathways on cerebral ischemia.
  • KO mice and wild-type mice are subjected to a permanent focal cerebral ischemia.
  • the parameters measured in order to determine the bearing of GRF1 on the ischemic damage result (i) from an examination of neurological function and (ii) from a histological quantification of the brain lesions.
  • mice Male mice weighing 22-37 g, derived from the C57B1/6 and 129SV genetic backgrounds, anesthetized with halothane at 1.4% in a nitrous oxide-oxygen mixture (70:30), are used. An incision is made between the eye and the left ear. The temporal muscle is folded back. A craniotomy is performed at the level of the temporal bone, which allows access to the middle cerebral artery. This is cauterized by electrocoagulation. A permanent focal cerebral ischemia is then caused by a left middle cerebral artery occlusion (MCA.O, (36)). During surgery, both the temperature of the temporal muscle and the body temperature are maintained at normal levels. The incision is then sutured, and the animals are put back in their cages in a room heated at 24-26° C.
  • MCA.O left middle cerebral artery occlusion
  • mice are divided into groups as follows:
  • a neurological examination is performed. During the examination, various points are scored: 0 (normal), 1 (flexing of the front leg), 2 (circular movement), 3 (flexing of the front leg and twisting of the thorax), 4 (loss of righting reflex).
  • mice reveal a significant neurological deficiency, in comparison to their own pre-ischemia neuronal score, 1 h and 24 h post-MCA.O.
  • the C57B1/6 mice no difference is observed between the wild-type mice and the KO mice in all of the points examined at the time.
  • the 129SV-KO mice exhibit a clearly less pronounced deficiency 1 h (p ⁇ 0.01) and 24 h (p ⁇ 0.05) post-ischemia.
  • mice after having had their “neuroscore” noted, the mice are sacrificed and the brains are rapidly removed and frozen in liquid isopentane at ⁇ 30° C. Coronal sections 40 ⁇ m thick are cut at various stereotaxic levels with a cryostat, and are then stained with 0.5% cresyl violet. The areas of lesion are measured with an image analyzer (Leica Q500) at various coronal levels. The volume of the brain lesions is then calculated by integration of the surface areas.
  • the decrease in the lesions in specific coronal levels might correspond to the structures involved in the motor and sensory-motor functions examined (in particular the neocortex and the cerebellum), in which the grf1 gene might be highly expressed (12). However, no effect is noted in the mice with a C57B1/6 genetic background.
  • the GRF1 protein might activate, in part, an apoptotic pathway responsible for vulnerability to ischemia. These results also suggest that the GRF1 protein itself might represent a target in particular for acute neurodegenerative disorders.
  • mice carrying the inactivating mutation for the grf1 gene show no characteristics describing obesity. The majority of them have no adipose tissue between the skin and the abdominal wall. In females, the deposit of fat around the uterine horns is very small and comparable in amount to that of 6-8-week-old wild-type animals.
  • the adult grf1 KO mice are also characterized by a lack of fat in the peritoneum and around the heart.
  • the assayed plasma leptin in the wild-type and mutant mice In order to correlate the observations made in the grf1 KO mice and wild-type mice with known characteristics of the obese phenotype, the assayed plasma leptin in the wild-type and mutant mice.
  • Leptin is a cytokine whose effect is to decrease food intake and to increase the expending of energy.
  • the relationship of proportionality exists between, firstly, the number of adipocytes—which secrete leptin—and the size of the lipid vesicles which they contain and, secondly the plasma concentration of this hormone (20).
  • the present inventors have determined the weight and the leptinemia of one-year-old male and female wild-type mice or mice mutant (KO) for the grf1 gene.
  • the group of animals studied consists of several sibships. Each sibship is composed of wild-type and mutant animals.
  • the blood samples were taken at the time of euthanasia of the animals by decapitation.
  • the plasma leptin was assayed using a radioimmunoassay (RIA) manufactured by the company Linco, in the form of a kit (ref: ML-82K) which is marketed in France by the company Clinisciences.
  • RIA radioimmunoassay
  • the first measurements of food consumption in the mutant mice indicate that food intake is decreased compared to the controls. However, if the amount of food absorbed is related back to the weight of the animals, there is no significant difference between the KOs and the wild-types.
  • GRF1 Protein for Screening Compounds by Measuring Serine/Threonine or Tyrosine Phosphorylation of the GRF1 Protein
  • This experiment is carried out on cells in culture.
  • GRF1 phosphorylation is induced in the cells in culture in various ways.
  • the GRF1 protein is then immunoprecipitated using an anti-GRF1 antibody or anti-tag antibody (10) (epitope recognized by monoclonal antibody and allowing detection or immunoprecipitation of the tagged protein, such as FLAG, myc or hemagglutinin (HA)), and the immunoprecipitate is then separated on an SDS-4-20% polyacrylamide gel.
  • the radioactivity of the band corresponding to the GRF1 protein (140 kDa) is quantified using a radiation detector of the PhosphorImager type.
  • the effectiveness of a molecule is calculated by the percentage inhibition of phosphorylation of the GRF1 protein, considering the value found for the nonstimulated cells to be the value 0% and the value found for the cells stimulated in the absence of the screened molecule to be the value 100%.
  • the first alternative to this method consists in incubating the cell lyzate previously obtained, in wells of CYTOSTAR plates (marketed by the company Amersham) precoated with an anti-GRF1 or anti-tag antibody in order to retain the GRF1 protein therein. After rinsing, quantification of the radioactive signal is obtained using a scintillation counter.
  • a second alternative to this method consists in treating the cells as above, but in the absence of labeled orthophosphate.
  • the cell lyzates are then used according to the ELISA assaying method.
  • the wells of the plates are coated with an anti-GRF1 or anti-tag antibody and the GRF1 phosphorylation is revealed and quantified with a second antibody which may or may not be labeled (radioelement, fluorescence, enzyme, etc.), and which may be either an anti-phosphotyrosine or an anti-phosphoserine/phosphothreonine.
  • the revelation and quantification are carried out using a third anti-species antibody directed against the second and labeled.
  • the signal is quantified using the radioactivity counter or a fluorescence spectrophotometer, or by measuring an enzyme activity, which leads to reading of optical density by spectrophotometry (plate readers).
  • the cells in culture may be:
  • the phosphorylation of the GRF1 protein in the cells in culture is obtained in the neuronal cells by adding 100 ⁇ M of carbachol (5 to 30 minutes) to the culture medium of the neurons.
  • GRF1 Protein for Screening Compounds by Measuring the Exchange Activity on the Ras Protein or the Rac or Cdc42 Proteins in the Presence of GRF1
  • the recombinant Ras, Rac or Cdc42 proteins are incubated in the presence of 3 H-GDP (0.5 ⁇ M final concentration, NEN, 111 ⁇ mol, 9 Ci/mmol) in the exchange buffer (50 mM Tris-HCl, pH 7.5, 1 mM MgCl 2 , 10 mM dithiothreitol, 1 mM EDTA, 1 mg/ml bovine serum albumin) for 30 minutes at 30° C., and then kept in ice.
  • 3 H-GDP 0.5 ⁇ M final concentration, NEN, 111 ⁇ mol, 9 Ci/mmol
  • the exchange buffer 50 mM Tris-HCl, pH 7.5, 1 mM MgCl 2 , 10 mM dithiothreitol, 1 mM EDTA, 1 mg/ml bovine serum albumin
  • MgCl 2 is then added to have a final concentration of 10 mM, in order to block the exchange reaction, and then 50 ⁇ l of each incubation are removed (Ras/Rac/Cdc42 bound to tritiated GDP) and are distributed in one of the 96 wells of a microplate.
  • the exchange reaction is started by addition to the wells of 10 ⁇ l of 10 mM cold GTP (0.1 mM final concentration), of recombinant GRF1 protein (all or parts) and of the test molecules.
  • a 40 ⁇ l aliquot is removed and filtered through a 0.45 ⁇ m nitrocellulose membrane (96-well plate with bottoms covered with a filtration membrane able to retain the proteins, sartorius SM 11306).
  • the radioactivity (tritiated GDP bound to small protein G) is counted in a scintillation counter, in the presence of scintillation fluid. In this case, it is the radioactivity which is then retained on the nylon filtration membrane which is counted.
  • An alternative to the filtration reaction consists in passing the contents of each well, after incubation, over a PD10 column preequilibrated with elution buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 1 mM PMSF, 5 mM DTT and 1 mM EDTA). In this case, it is the radioactivity of the eluate from the column which is measured (4 ml of elution buffer passed over the column+10 ml of Beckman ReadyGel scintillation fluid).
  • elution buffer 50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 1 mM PMSF, 5 mM DTT and 1 mM EDTA.
  • the exchange activity is measured by the difference in amount of radioactivity in the presence of GRF1 protein compared to that obtained in an assay without GRF1 protein.
  • the effectiveness of a molecule is calculated by the inhibition of this exchange activity.
  • the recombinant Ras, Rac or Cdc42 proteins may be wild-type Ras, Cdc42 or Rac proteins of various origins (human, murine, etc.) expressed:
  • the recombinant GRF1 proteins may be whole or fractions of GRF1 proteins of various origins (human, murine, etc.) containing the exchange domains, either CDC25 or DH, or both, expressed in baculoviruses or in E. coli , and purified on an affinity column.
  • a variant of the preceding technique consists in using the ability either of Ras-GTP to bind to Raf-1 via the Ras-Binding domain (RBD) of Raf, or of Rac1-GTP or Cdc42-GTP to bind to PAK1 via the CRIB domain of the latter (39).
  • the SPA (Scintillation Proximity Assay) system described in U.S. Pat. No. 4,568,649, EP 154 734 and JP 84/52452, makes it possible to demonstrate and quantify the binding between two proteins in a high throughput screen.
  • the subsequent steps consist in adding MgCl 2 to a final concentration of 10 mM in order to block the exchange reaction, and then in removing 50 ⁇ l of each incubation and distributing them in one of the 96 wells on a microplate.
  • the exchange reaction is started by addition to the wells of 10 ⁇ l of tritiated GTP (NEN 111 ⁇ mol, 9 Ci/mmol), of the recombinant GRF1 protein and of the test molecules.
  • GST-Raf (RBD domain) or GST-PAK (CRIB domain) proteins at the concentration of 0.0013 mg/ml in a 50 mM Tris-HCl buffer, pH 7.5, containing 2 mM dithiothreitol, and 40 ⁇ l of a mixture of anti-GST immunoglobulin (0.12 mg/ml) and SPA protein A PVT coupled to microspheres (Amersham; 12.6 mg/ml) in a 50 mM Tris-HCl buffer, pH 7.5, containing 2 mM dithiothreitol and 2 mM MgCl 2 , are added. The plates are then sealed, agitated for 1 h at 22° C., and then centrifuged at 760 g for 2 minutes and counted in a scintillation counter.
  • the effectiveness of the molecule is calculated by the percentage inhibition of the exchange activity and is equal to: 100 ⁇ (1 ⁇ (value in the presence of GRF1 and of the molecule ⁇ control in the absence of GRF1 and of the molecule)/(value in the presence of GRF1 and in the absence of the molecule ⁇ control in the absence of GRF1 and of the molecule)).
  • yeast Saccharomyces cerevisiae
  • competition systems producing a physiological disorder
  • double-hybrid system All the techniques and methods which use yeast are widely described in the literature (40-45).
  • yeast RAS proteins are homologous to the mammalian Ras proteins (the latter complement the inactivating mutations of RAS proteins in yeast) and the S.c. CDC42 protein is homologous to the mammalian Rac proteins.
  • S.c. CDC42 protein is homologous to the mammalian Rac proteins.
  • exchange factors CDC25 and SDC25 are homologous to GRF1 and their catalytic portions are active on the mammalian Ras proteins (46, 47).
  • GRF1 by virtue of its domain homologous to CDC25, is capable of complementing any inactivating mutation of CDC25, or else of a double mutant CDC25/SDC25 (the SDC25 mutants or KOs having no phenotype).
  • An exchange factor, called CDC24, which can be complemented by the DH domain of Vav is also known for S.c. CDC42 (48). It is possible to extrapolate an exchange activity for all or part (including the DH domain) of GRF1 on CDC42 in a yeast mutant for CDC24, these inactivating mutations having a lethal phenotype. This activity of GRF1 (all or part) would totally or partially (depending on its affinity) mimic the effect of CDC24.
  • GRF1 (all or part) is capable of mimicking the function of the yeast exchange factors (CDC25, CDC24) when they are inactivated by a mutation leading to a phenotype of the growth arrest type. The effect of this complementation is to allow survival of the yeast by restoring growth.
  • the specificity of the method of action of the molecule on the RAS/CDC25 or CDC42 domain/DH domain system will be verified by measuring the action of the molecule on a wild-type yeast (information regarding the antifungal activity).
  • the screening will be carried out, for small molecules, in a yeast permeabilized by techniques known to those skilled in the art, consisting in raising the intracellular concentration of the molecules by adjusting the membrane permeability and the expression of genes involved in detoxification processes (for example using mutants of the Erg6 gene and/or of the genes of the PDR family).
  • permeabilization techniques are described in the following patent applications: FR 9411509 and WO 96/1082.
  • a strain of the genus S. cerevisiae , CDC25 TS or CDC24 TS (thermosensitive mutations), is used. It can only grow under permissive temperature conditions. It is grown on the following culture medium:
  • This medium can be solidified by adding 20 g/l of agar (Difco).
  • auxotrophic yeasts In order to allow the growth of auxotrophic yeasts on this medium, it is necessary to add thereto the nitrogenous bases or amino acids on which they are dependant, at 50 mg/ml. 100 ⁇ g/ml of ampicillin are added to the medium in order to avoid bacterial contamination.
  • the Escherichia coli strain TG1 having the genotype supE,hsd ⁇ 5,thi, ⁇ (lac-proAB),F′[traD36 pro A + B + lacI q lacZ ⁇ M15], is used to construct plasmids and to amplify and isolate plasmids. It is grown on the following medium:
  • This medium can be solidified by adding 20 g/l of agar (Difco).
  • Ampicillin at 100 ⁇ g/ml is used to select the bacteria which have received the plasmids carrying as a marker the gene for resistance to this antibiotic.
  • the yeast are made competent by treatment with LiAC/PEG according to the method described by Gietz et al. (49) and transformed with 1 ⁇ g of plasmid allowing constitutive or inducible expression of all or part of GRF1 in yeast. After the transformation steps, the yeasts are put back in culture under nonpermissive conditions. The clones selected will be those which have been complemented by GRF1.
  • a selected clone complemented by GRF1 is plated out on dishes containing the selection medium onto which drops of chemical compounds are applied.
  • the dishes are placed under permissive conditions.
  • the products giving a ring of yeast growth inhibition are selected and tested on a control wild-type yeast in order to eliminate the molecules with antifungal activity which give the same type of growth inhibition.
  • GRF1 the expression of all or part of GRF1 in the yeast is accompanied by a growth disturbance due to the constitutive stimulation of the exchange on the RAS or CDC42 proteins.
  • a molecule which is active on the system will lead to a more or less complete standardization of the physiological disorder (phenotype) engendered by the expression of GRF1.
  • a wild-type yeast strain of the genus S. cerevisiae is used. It is grown on the minimum YNB medium as described in 2.1. The construction and the amplification of the plasmids are carried out as described in 2.1.
  • GRF1 the expression of GRF1 leads to the overactivation of yeast RAS. Such yeasts will then no longer be capable of producing glycogen stores when they reach the prestationary growth phase. They will be identified by the absence of brown coloration after exposure to iodine vapor.
  • the screening will be carried out in a permeabilized yeast according to the techniques described above.
  • Drops of chemical compounds are applied directly to the surface of culture dishes seeded beforehand with the yeast expressing all or part of GRF1.
  • the dishes are left to grow and then exposed to iodine vapor in a closed chamber containing iodine crystals.
  • the products which allow yeast growth and which restore the appearance of the brown coloration will be selected as positive.
  • GRF1 GRF1
  • a wild-type strain of the genus S. cerevisiae is used. It is grown on the minimum YNB medium as described in 2.1. The construction and the amplification of the plasmids are carried out as described in 2.1.
  • the wild-type yeast is made competent by treatment with LiAC/PEG according to the method described by Gietz et al. (49) and transformed with 1 ⁇ g of plasmid allowing inducible expression of all or part of GRF1 in yeast (example: the expression of the grf1 gene is placed under the control of the Gal 4 gene promoter which is inducible in the presence of galactose as sole carbon source in the culture medium).
  • GRF1 leads to a disturbance in the cell signaling which uses the RAS proteins in the yeast, and results in a phenotpye of the growth arrest type.
  • the screening will be carried out in a permeabilized yeast according to the techniques described above.
  • Drops of chemical compounds are applied directly to the surface of culture dishes seeded with a yeast transformed with the plasmid for inducible expression of GRF1, but cultured beforehand in the absence of inducing agent.
  • the dishes are incubated under conditions which allow GRF1 expression.
  • the products which allow growth of the yeast will be selected as positive.
  • the technique is based on the preparation of a first fusion protein between Ras, CDC42Hs or Rac and a DNA-interacting domain, and of a second fusion protein between GRF1 (all or CDC25-like or DH domains) and a transactivating domain. Interaction between the two hybrid proteins leads to activation of a reporter gene (HIS3, LEU2, URA3, CYH2, CAN1, LacZ, GFP, etc.).
  • the system is used in the appropriate yeast (example: mutant ura3 if the reporter gene is URA3).
  • the CL9 yeast strain is used as a tool for screening molecules which interfere in the double-hybrid system. It makes it possible to demonstrate protein-protein interaction. It can be made permeable by introducing mutations in the family of PDR genes and of the ERG6 gene involved in detoxification processes.
  • the Escherichia coli strain TG1 having the genotype supE, hsd ⁇ 5,thi, ⁇ (lac-proAB),F′[traD36 pro A + B + lacI q lacZ ⁇ M15], is used to construct plasmids and to amplify and isolate plasmids.
  • the plasmids used are as follows:
  • the vector pGAD10 supplied by Clontech® allows expression in yeast of a fusion protein between all or part of GRF1, and the transactivating domain of GAL4 (GRF1-TA protein).
  • the vector pGBT9 provided by Clontech®, allows expression in yeast of a fusion protein between Ras, Rac or Cdc42 and the DNA-interacting domain of GAL4 (Ras-BD or Rac-BD or Cdc42-BD proteins).
  • the carboxy-terminal domain for farnesylation of Ras or for geranylgeranylation of the Rac and Cdc42 proteins are removed from the construct. This makes it possible to obtain proteins which do not attach to the lipid membranes and which enter the cell nucleus efficiently.
  • the CL9 yeast is made competent by treatment with LiAC/PEG according to the method described by Gietz et al. (49). It is then transformed with 1 ⁇ g of each of the plasmids allowing expression of the fusion proteins, which make up the double-hybrid system. Expression of these fusion proteins leads to the strain being sensitive to cycloheximide.
  • a product which interferes with the interaction between the fusion proteins of the double-hybrid system will allow yeast growth on this type of medium.
  • the yeast is plated out on the surface of a selective medium containing 10 ⁇ g/ml of cycloheximide. Drops of chemical compounds are applied directly to the surface of the dish. The products selected are those which give a halo of growth around the deposit.
  • the screening will be carried out in a permeabilized yeast according to the techniques described above.
  • inhibition of the protein-protein interaction by active molecules can be revealed by the decrease in expression of a reporter gene. This can be demonstrated, as appropriate, using a colorimetric, fluorimetric or enzyme (example: ⁇ -galactosidase) assay. Growth inhibition can be measured using a selective medium (for example: fluorate medium for the use of the URA3 reporter gene or canavanin medium for CAN1).
  • a selective medium for example: fluorate medium for the use of the URA3 reporter gene or canavanin medium for CAN1.
  • ⁇ -Galactosidase activity is measured in the following way:
  • the L40 strain of the genus S. cerevisiae (Mata, his3D200, trp1-901, leu2-3,112, ade2, LYS2:: (lexAop)4-HIS3, URA3::lexAop)8-LacZ, GAL4, GAL80) is used as a screening tool.
  • This strain makes it possible to demonstrate protein-protein interaction when one of the protein partners is fused to the LexA protein (70). It was grown on the minimum YNB culture medium.
  • the Escherichia coli strain TG1 having the genotype supE, hsdD5,thi,D(lac-proAB),F′[traD36 pro A + B + lacI q lacZDM15], was used to construct plasmids and to amplify and isolate plasmids (as described in 2.1).
  • the plasmids used are as follows:
  • the vector pGAD10 provided by Clontech®, allows expression in yeast of fusion proteins between all or part of GRF1, and the transactivating domain of GAL4.
  • the vector pLex9 (pBTM116) allows expression in yeast of fusion proteins between Ras, Rac or Cdc42 and the DNA-interacting domain of the LexA protein.
  • the yeast is made competent by treatment with LiAC/PEG according to the method described by Gietz et al. (49). It is then transformed with 1 ⁇ g of each of the plasmids allowing expression of the fusion proteins, which make up the double-hybrid system. Expression of these proteins leads to expression of ⁇ -galactosidase.
  • a sheet of nitrocellulose is placed over the Petri dish containing the layer of yeast and the drops of chemical compounds to be screened. This sheet is then immersed in liquid nitrogen for 30 seconds in order to rupture the yeast and to thus release the ⁇ -galactosidase activity.
  • the sheet of nitrocellulose is placed, colonies facing upwards, in another Petri dish containing a Whatman paper presoaked with 1.5 ml of PBS solution (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , pH7) containing 15 ⁇ l of X-Gal (5-bromo-4-chloro-3-indoyl- ⁇ -D-galactoside) at 40 mg/ml in N,N-dimethylformamide.
  • PBS solution 60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , pH7
  • X-Gal 5-bromo-4-chloro-3-indoyl- ⁇ -D-galactoside
  • the carboxy-terminal domain for farnesylation of Ras or for geranylgeranylation of the Rac and Cdc42 proteins can be removed from the construct, which makes it possible to obtain proteins which do not attach to the lipid membranes and which enter the cell nucleus efficiently.
  • GRF1 Activation of small Gs by GRF1 would induce their loading with GTP and therefore their interaction with their effector (PAK1 or Raf-1), and would therefore allow expression of the reporter gene as described above.
  • the CL9 yeast strain is used as a tool for screening molecules which interfere in the double-hybrid system. It makes it possible to demonstrate protein-protein interactions. It can be made permeable by introducing mutations in the family of PDR genes and of the ERG6 gene involved in detoxification processes. It was grown on the minimum YNB medium.
  • the Escherichia coli strain TG1 having the genotype supE, hsd ⁇ 5,thi, ⁇ (lac-proAB),F′[traD36 pro A + B + lacI q lacZ ⁇ M15], is used to construct plasmids and to amplify and isolate plasmids. It is grown on LB medium.
  • the plasmids used are as follows:
  • the vector pGAD10 supplied by Clontech®, allows expression in yeast of a fusion protein between PAK1 (CRIB domain) or c-Raf1 (RBD domain) and the transactivating domain of GAL4 (Pak1-TA or c-Raf1-TA protein).
  • the vector pGBT9 supplied by Clontech®, allows expression in yeast of a fusion protein between Ras, Rac or Cdc42 and the DNA-interacting domain of GAL4 (Ras-BD or Rac-BD or Cdc42-BD proteins).
  • a vector which allows expression in yeast of GRF1 (all or parts).
  • the CL9 yeast is made competent by treatment with LiAC/PEG according to the method described by Gietz et al. (49). It is then transformed with 1 ⁇ g of each of the plasmids allowing expression of the fusion proteins and GRF1, which make up the double-hybrid plus one system. Expression of these proteins leads to the strain being sensitive to cycloheximide.
  • a product which interferes with the activation by GRF1 of the Ras-BD, Rac-BD or Cdc42-BD fusion proteins of the system will allow yeast growth on this type of medium.
  • the yeast is plated out over the surface of a selected medium containing 10 ⁇ g/ml of cycloheximide. Drops of chemical compounds are applied directly to the surface of the dish.
  • the products selected are those which give a halo of growth around the deposit.
  • the screening is carried out in the yeast permeabilized according to the techniques described above.
  • the active molecules By inhibiting the protein-protein interaction, the active molecules can be revealed by the decrease in expression of the reporter gene. This decrease can be demonstrated, as appropriate, using a calorimetric, fluorimetric or enzyme assay. Growth inhibition can be measured using a selective medium (for example: fluorate medium for the use of the URA3 reporter gene or canavanin medium for CAN1).
  • a selective medium for example: fluorate medium for the use of the URA3 reporter gene or canavanin medium for CAN1.
  • GRF1 Protein for Screening Compounds by Measuring the Interaction between GRF1 and the Beta-Gamma Subunits of Heterotrimeric G Proteins
  • the double-hybrid system in yeast appears to be the appropriate tool for carrying out a screen to reveal molecules capable of disturbing this interaction. It might use either the whole GRF1 protein, or the PH domains, or the series of PH-DH-PH domains as found naturally in the GRF1 protein.
  • the screening assay for GRF1 inhibitors uses stable clones of cells transfected with grf1, for which the present inventors have been able to show over-expression of the transgene by western blotting.
  • GRF1 Overexpression of GRF1 imparts on the cells the ability to be cloned in agar, which is verified according to the following protocol:
  • the cells are counted and resuspended in complete medium without phenol red, at a concentration of 2.5 ⁇ 10 5 cells per ml.
  • top layer prepared from the following mixture:
  • test molecules are diluted in 1 ⁇ medium at suitable concentrations, and 75 ⁇ l are added to the layers of agar in the wells.
  • the cells are counted after two weeks by staining with calcein:
  • Calcein AM (molecular probes C-1430, 4 mM DMSO) is diluted to 10 ⁇ M in HBSS and stored at ⁇ 20° C. 25 ⁇ l of calcein AM are added to each well and the cells are left in the incubator (37° C.) for at least one hour. The plate is read in a C0 device.
  • a GRF1 inhibitor will be revealed by the decrease in the number of clones in agar.
  • Any immortalized mammalian cell transfected with a eukaryotic expression vector (plasmid) allowing expression of all or part (CDC25-like domain or DH domain) of human or murine grf1 can also be used.
  • This expression vector at the same time imparts the phenotype resistant to a selection agent such as hygromycin, neomycin, zeocin or the like, and as such makes it possible to select and then clone (by FACS or by limiting dilution) the cells transfected and expressing the transgene.

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