FR2845604A1 - Use of cDNA encoding a mutant 5-HT4 receptor for treatment or prevention of diseases associated with dysfunctional synthesis of cyclic adenosine monophosphate, e.g. neurodegeneration or asthma - Google Patents

Abstract

Using cDNA (I) that encodes the receptor 5-HT4-D100A (II) to prepare a composition for treatment and/or prevention of a disease caused by a dysfunction in synthesis of cyclic adenosine monophosphate (cAMP), is new. In (II), the Asp-encoding codon 100 of transmembrane domain III is replaced by an Ala-encoding codon.

Description

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 PHARMACEUTICAL COMPOSITION COMPRISING A 5-HT4D100A RECEPTOR, AND THERAPEUTIC USES THEREOF

 The present invention belongs to the field of pathologies related to failures in the intracellular synthesis of cyclic adenosine monophosphate (cAMP), and in particular to the use of pharmaceutical compositions useful in the prevention and / or treatment of pathologies related to a failure in cAMP synthesis, particularly neurodegenerative pathologies and respiratory pathologies.

 Among the neurodegenerative diseases that have a significant impact on human health, Parkinson's disease, Alzheimer's disease, or Huntington's chorea are known, among others.

 Parkinson's disease is a neurodegenerative disease that results in difficulties triggering voluntary movement. This disease is due to the degeneration of dopaminergic neurons located in the substantia nigra. Alzheimer's disease is a progressive neurodegenerative disease of the brain that causes senility and dementia. The accumulation of the beta-amyloid protein is at the origin of the appearance of the first symptoms of this disease. Huntington's chorea, which is characterized by a movement disorder associated with progressive dementia, is due to the alteration of the striatal neurons.

 Most neurodegenerative diseases are diagnosed only at the onset of symptoms, at a time when neuronal degeneration is already very

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 advanced, the treatments are and will be for a long time symptomatic.

 In the case of Parkinson's disease, the treatments consist of administering L-dopa, a precursor of dopamine, to increase its synthesis in neurons still alive. These treatments have adverse effects (dyskinesias) that occur after a few years. In the most severe cases, the surgical placement of high frequency stimulation equipment of the subtalamic nucleus can be used.

 In the case of Alzheimer's disease, pharmacological treatments are ongoing according to three approaches that affect the cholinergic system.

Administer precursors of acetylcholine, such as lecithin-choline. Administering drugs that increase the release of acetylcholine such as hydergine, administering cholinesterase inhibitors such as tacrine and velnacrine, however, the clinical benefits generally remain modest with many adverse effects.

 Huntington's chorea treatments use drugs that reduce dopaminergic activity. Their main undesirable effects are agitation and parkinsonism.

 None of these treatments is satisfactory from a therapeutic point of view because, in any case, these processes can not be reversed by the current treatments mentioned above.

 Existing treatments that are only symptomatic do not in any way prevent neuronal degeneration, they are only intended to further stimulate the few neurons in temporary survival. The outcome by these treatments is of any

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 fatal way, only the prolongation of some reinforced neuronal functions is possible by the current treatments.

 Thus, to overcome these drawbacks, the Applicant has developed a novel therapeutic composition capable of locally increasing the intracellular concentration of a protective compound: cAMP.

 In the context of neurodegenerative pathologies, thanks to its neuroprotective effect, cAMP can save dying neurons from death. It thus avoids the programmed death of the neuron and consequently makes it possible to maintain and / or recover partially or totally its activity. As a result, the disease will be treated at its origins. The regions of the brain involved are those where the neurons degenerate.

 It is known that, in addition to the neurodegenerative diseases mentioned above, other pathologies, such as respiratory diseases, and in particular asthma and cystic fibrosis, are related to an abnormality in the synthesis of cAMP.

 It is now recognized that asthma is an inflammatory disease hence the importance of anti-inflammatory drugs in the daily treatment of asthma and for curing acute attacks. Numerous basic research in this field has demonstrated that inflammation processes can be regulated by cAMP via inhibition of phosphodiesterases in order to increase the intracellular concentration of cAMP (Trifilieff, A. et al., J. Pharmacol. Exp.Ther. (2002) 301: 241-248) or via activation of beta2-adrenergic receptors positively coupled to adenylate cyclase

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 (Aksoy MO, J Allergy Clin Immunol (2002) 109: 491-497, Gao H. et al., Chin Med J (2001) 114: 1317-1319). In both studies, it has been clearly demonstrated that increasing the intracellular cAMP concentration leads to beneficial effects in the treatment of asthma. CAMP is considered a bronchodilator to decrease the inflammatory response in the case of asthma but also the case of cystic fibrosis where it is known that the gene of the protein cystic fibrosis transmembrane conductance regulator or (CFTR) codes for a a protein whose function is to be a cAMP regulated chlorine channel, (Andersson, C. et al.

 Clin Sci (2002) 103: 417-424; Gen. Physiol. (2002) 120: 407-418; Bebok Z. J. Biol. Chem. (2002) in press).

The local increase in cAMP concentration is obtained, within the framework of the present invention, by means of a pharmaceutical composition inducing a local and specific increase in the expression, in a targeted tissue, of a receptor. HT4 mutated, so that it is activated by a synthetic ligand but not by its natural ligand
Said receptor is then activated only by specific synthetic ligands to locally induce the synthesis of cAMP protecting the cells and in particular neuronal cells and epithelial cells.

 The case of dopaminergic neurons of the substantia nigra is known, in the case of Parkinson's disease, for example (Jellinger, K. (1990) Adv Neurol 53: 1-16). Several beneficial effects of cAMP production by the 5-HT4-D100A receptor in dopaminergic neurons are expected: increased neuronal survival 2) transcriptional stimulation

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 coding for tyrosine hydroxylase (thus an increase in dopamine synthesis) 3) dopamine stimulation 4) an anti-inflammatory effect.

 In the case of Alzheimer's disease, a local increase of cAMP would promote the secretion of the soluble form of the precursor of amyloid beta protein, a non-amyloidogenic form (Robert, SJ, Zugaza, JL, Fischmeister, R., Gardier, AM, and Lezoualc'h, F. (2001) J Biol Chem 276: In the case of Huntington's Chorea a local increase in cAMP would save the putamen neurons from programmed death (Mantamadiotis, T., Lemberger, T., Bleckmann, SC, Kern, H., Kretz, O., Martin Villalba, A., Tronche, F., Kellendonk, C., Gau, D., Kapfhammer, J., Otto, C., Schmid, W., and Schutz, G.

(2002) Nat Genet 31: 47-54. ).

 The pharmaceutical composition of the invention also opens the way to a novel form of gene therapy for the treatment of diseases requiring regulation of intracellular cAMP concentration using a Galpha-coupled protein receptor or a mutated RASSL 5-HT4. as a means of regulating the intracellular concentration of cAMP.

 Pharmaceutical compositions comprising said mutated 5-HT4-D100A mutant receptors constitute tools that modulate the transduction pathways and thus make it possible to correct pathologies related to cell survival, in particular neuronal, depending on the intracellular concentration of cAMP. .

 The neuroprotective effect of cAMP on the neurons of the central nervous system makes it possible to

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 is a new form of gene therapy that involves the expression of the cAMP-producing mutated receptor in brain regions where neurons are degenerating.

 In the three examples of neurodegenerative diseases mentioned above, increasing the intracellular cAMP concentration can strongly reduce neuronal degeneration (Jellinger, K.

(1990) Adv Neurol 53: 1-16; Robert, S.J., Zugaza, J.

L., Fischmeister, R., Gardier, A. M., and Lezoualc'h, F.

(2001) J. Biol. Chem. 276: 44881-44888; Mantamadiotis, T., Lemberger, T., Bleckmann, SC, Kern, H., Kretz, O., Martin Villalba, A., Tronche, F., Kellendonk, C., Gau, D., Kapfhammer, J., Otto, C., Schmid, W., and Schutz, G. (2002) Nat.Genet. 31: 47-54)
The object of the present invention is to reverse the cellular processes which, once switched on, lead to the development of neurodegenerative diseases.

 This novel therapeutic composition comprises a receptor which, advantageously, has been rendered silent to its endogenous ligand by a simple mutation, but which remains always activatable solely by exogenous ligands.

 The interest of these receptors called RASSL Receptor Activated Solely by Synthetic Ligands was first mentioned by Conklin in 1998 (Coward P. et al (1998) Proc Natl Acad Sci USA 95: 352-35). . Examples of receptors that by one or more mutations become completely silent in the presence of their natural agonist while keeping the

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 ability to be stimulated by synthetic agonists are still very rare. The only known example to date is the opiate kappa receptor, which is negatively coupled to adenylate cyclase. Thus, expression of this receptor expressed in the heart, has the ability to modulate the heart rate by decreasing, in this case, the intracellular concentration of cAMP (Redfern CH et al (2000) Proc Natl.Acad USA 97: In the context of the present invention is meant by: - Agonist: any molecule capable of generating by its binding to its receptors, a biological response similar to an endogenous mediator.

 - Antagonist: any molecule capable of blocking the action of agonists.

 Inverse agonist: An agonist capable of generating a reverse biological response to that of normal agonists.

 The present invention uses a particular silencing receptor, a 5-HT4 receptor in which the D100 aspartic acid occupying position 100 has been mutated to Alanine (5-HT4D100A). This mutated receptor no longer responds to the serotonin which is its endogenous ligand, but is still activatable by specific compounds of this receptor that cross the blood-brain barrier very well.

 This type of "RASSL" that the plaintiff obtained for the 5-HT4 receptor by a simple mutation is unique. Indeed, 5-HT4-D100A is the first "RASSL" that becomes totally non-activatable by its endogenous ligand, which is not the case of the opiate kappa receptor (Coward P. et al. (1998) Proc. Natl Acad.

Sci. USA 95: 352-35).

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 The 5-HT4-D100A receptor is totally stimulated by a very large number of drugs totally different in structure and bio stability.

 Certain highly selective 5-HT4 receptor compounds in the arylketone class behave as partial agonists of the native receptor and become total agonists of the mutated 5-HT4-D100A receptor. They also have the advantage of being very hydrophobic and very easily pass the blood-brain barrier.

 More specifically, the subject of the present invention is a pharmaceutical composition comprising a DNA sequence (double-stranded deoxyribonucleic acid) encoding a mutated 5-HT4-D100A receptor for the preparation of a medicament for the treatment and / or prevention of a pathology caused by dysfunction in cAMP synthesis.

 By way of example, among the DNAs which may be used in the therapeutic composition of the invention, mention may be made of the DNA whose sequence is identified by SEQ ID No. 1 in the attached sequence listing.

 This sequence is the mutated form of a wild-type 5-HT4 receptor (the sequence of which is identified under the number SEQ ID No. 3 in the attached sequence listing and whose N accession in the GENBANK sequence base is Y09586) where the GAC codon at position 297-300 coding for aspartic acid has been replaced by the GCT codon encoding alanine.

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la 5-HT lorsque l'aspartateloo, du domaine transmembranaire III, hautement conservé au cours de l'évolution, est muté en alanine. This type of receiver is unable to bind to

5-HT when the aspartateloo, transmembrane domain III, highly conserved during evolution, is mutated to alanine.

 Aspartate in the receptor binding pocket is an essential anchor for serotonin to bind to the receptor. The receptor remains, on the other hand, fully activatable by numerous synthetic agonists and specific for the 5-HT4 receptor, such as the benzimidazolone derivatives (BIMU-8), the benzamine derivatives (cisapride, renzapride) and benzoates (Ml 10302, ML 10375) or the arylketone derivatives (RS 66333) (Bockaert, J., Fagni, L., and Dumuis, A. (1997) in Handbook of Experimental Pharmacology: Serotoninergic Neurons and 5-HT Receptors in the CNS (Baumgarten, HG, and Gôthert, M ., eds) 129: 439-465, Spinger-Verlag, Berlin Heidelberg New York). These molecules have a very large structure and aspartate100 is not useful for their binding on the receptor.

 In the context of the present invention, the term "silent 5-HT4 receptor" is intended to mean any functional 5-HT4 receptor that is silent, that is to say which has the mutation of the aspartic residue of the transmembrane domain III of the corresponding wild-type receptor in alanine and retains its binding capacity for synthetic ligands.

 Thus, the pharmaceutical composition may comprise any cDNA sequence coding for 5-HT4D100A receptors of any species, in particular human, murine or rat, having an identity of at least 90% with said sequence SEQ ID No. : 1, and

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 in which the GCT / GAC mutation, described above, was introduced.

 It may be the receptor identified by the sequence SEQ ID No. 1 in the attached sequence listing, but also the pharmaceutical preparation of the invention may comprise cDNA sequences coding for truncated 5-HT4D100A receptors which retain their binding capacity for their synthetic ligands and in particular 5-HT4-D100A receptors in which the C-terminal domain is absent, or else 5-HT4-D100A receptors exhibiting one or more point mutations.

 Advantageously, these last truncated or mutated receptors are expressed constitutively.

 Such silent 5-HT4-D100A mutant receptors can be obtained by site-directed mutagenesis (Mialet, J et al., Br.J.Pharmacol (2000) 130: 527-38, Joubert L. et al., J.Biol.Chem. (2002) 277: 25502-25511).

 In order to express this mutated 5-HT4-D100A receptor, in regions of the brain where neurons degenerate, the use of transfer vectors, such as non-viral or viral vectors containing the coding cDNA sequence, can be envisaged. for said mutated 5-HT4-D100A receptor.

(Mol.Ther.(2002) 6(3):349 ; (Curr.Opin.Mol.Ther. Among the viral vectors, mention may be made of adenovirus-type vectors (Eur cytokine Netw 2002; 13 (3): 324-30, vectors derived from viruses associated with adenovirus type 2 (AAV) (J Virol 2002 Jan; 76 (2): 791-801) (Curr.Opin.Mol.Ther 2002: 4 (4): 349-58), lentivirus vectors with different pseudo-types

(Mol.Ther. (2002) 6 (3): 349; (Curr.Opin.Mol.Ther.

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 (2002) 4 (4): 349-58; (Basic.Res., Cardiol. (2002) 97 (5): 348-58), vectors derived from feline immunodeficiency viruses (J.Virol. (2002); 76 (19): 9624-34); vectors derived from Foamy virus (FV) (Mol Ther (2002) 6 (3): 321).

 These viral vectors are free of any pathogenic and toxic effects. They are particularly effective for transfecting neurons and protein expression is very stable.

 These vectors are used with suitable promoters, depending on the target tissue. For neuronal cells of the substantia nigra or dopaminergic neurons, it is the promoter of the dopamine transporter.

For targeted transfection of other tissues, specific promoters can be used, for example for HIV-like vectors and targeted transfection into neurons the promoters described in: 272: (1996); PNAS 93: 11382 (1996); J. Virol. 71: 6641 (1997); Hmm. Gene Ther. 11: 179 (2000); Nature Biotech. 19: 446 (2001); Hmm. Gene Ther. 13: (2002)
For transfection of airway epithelial cells with HIV-like viral vectors, suitable promoters are indicated in the following publications: Hum. Gene Ther. 8: (1997); J. Virol. 75: (2001).

 Other examples of promoters that can be used in the context of the present invention are also those cited in: Blood 95: 2499 (2000) and in Gene Ther. 7:20 (2000).

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vecteur PAAV-CAG-5-HT4-DlooA-HA est construit dans une cassette contenant un promoteur fort (P-actine/enhancer cytomégalovirus ou promoteur CAG) selon le protocole décrit par Kapplit et al. (Kaplitt, M. G., Leone, P., Samulski, R. J., Xiao, X., Pfaff, D. W., O'Malley, K. For example, according to a particular mode of implementation, in the therapeutic composition of the invention, the plasmid contains the complete genome of AAV (psub201) and the packaging plasmid (pAAV / Ad). The

PAAV-CAG-5-HT4-DlooA-HA vector is constructed in a cassette containing a strong promoter (P-actin / cytomegalovirus enhancer or CAG promoter) according to the protocol described by Kapplit et al. (Kaplitt, MG, Leone, P., Samulski, RJ, Xiao, X., Pfaff, DW, O'Malley, K.

L., and During, M.J. (1994) Nat Genet 8 (2), 148-54. ).

According to another embodiment, the pharmaceutical compositions for the preparation of a medicament for the treatment of a respiratory disease will comprise the DNA of the 5-HT4-D100A receptor introduced into a recombinant vector of the adenovirus-associated virus (AAV) or lentivirus placed behind a cassette containing a strong CMV promoter and a tissue-specific promoter in which it is desired to express said receptor, as an example of such a promoter, mention may be made of Cytokeratin K18 (Lusky M. and J Virol (1999) 73: 8308-19)
Advantageously, the pharmaceutical composition of the invention will be administered by injection. In particular, the patients will be injected with the viral particles comprising the nucleic acid encoding the 5-HT4D100A receptor, under stereotactic control, for the treatment of neurodegenerative diseases this injection may be performed at the level of the brain areas in successive injections, which may be from 2 to 10, preferably from 3 to 5, spaced 24 hours each.

 Synthetic agonists of the 5-HT4 receptor will then be administered at defined times

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 previously. These compounds cross the blood-brain barrier, are very active and exhibit high bio-stability in vivo.

 An increase in the intracellular cAMP concentration can be obtained by activation of the mutated 5-HT4-D100A receptor, expressed specifically, by means of the pharmaceutical composition of the invention, in the dopaminergic neurons of the substantia nigra, in neurons. hippocampus or neurons of the striatum according to the protocols described in the examples below.

 This activation of the mutated 5-HT4-D100A receptor may be carried out, for example, with synthetic agonists, examples of which include BIMU8 and RS 66333 compounds, when the first signs appear. clinical disease.

 Other 5-HT4-D100A receptor agonists that do not bind to the native receptor can also be used.

 Another subject of the invention relates to a method for screening ligands of the mutated 5HT4 receptor.

 Such a screening method is used, for example in vitro, it comprises bringing said receptor into contact with said chemical compound, under conditions permitting their binding, followed by the detection of this bond.

 It can be the isolated, purified or partially purified receptor comprising the fragment of the sequence of the receptor containing the D100 mutation, fixed on solid supports, according to techniques well known to those skilled in the art, for example and

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 non-limiting, one can quote tests by competition, tests by means of biosensors or by means of membrane fragments extracted from cells expressing said receiver.

 Such a screening method may also be carried out in vivo on isolated cells genetically modified to express said 5HT4D100A receptor. This method then comprises contacting genetically modified cells expressing said mutated 5-HT4D100A receptor with said chemical compound, under conditions permitting their binding and the detection of the activity of said receptor.

 This latter method of screening is particularly suitable for the identification of agonist compounds of the 5-HT4D100A receptor. In this particular embodiment, isolated cells, genetically modified to express the 5-HT4D100A receptor, are contacted with said chemical compound to be tested, under conditions allowing the activation of said receptor, and a possible increase is detected. the activity of said receiver.

 Likewise, this screening method is particularly suitable for the identification of antagonist compounds of the 5-HT4D100A receptor. In this particular embodiment, isolated cells, genetically modified to bring said 5-HT4D100A receptor into contact with said chemical compound to be tested, are brought into contact under conditions allowing the activation of said receptor and a possible decrease in activity of said receiver.

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 Finally, the subject of the invention is also a solid support on which is fixed a 5-HT4D100A receptor according to the invention or a functional fragment thereof.

 According to a particular mode of implementation, on the solid support of the invention is fixed a 5-HT4D100A receptor whose polypeptide sequence is identified in the appendix under the number SEQ ID No: 2.

 According to a preferred embodiment, the receptor is fixed on said solid support by covalent or non-covalent bonds.

 Preferably, the solid support according to the invention is selected from the group of supports comprising inorganic supports such as metal, glass, silicon and organic supports based on polymers or co-polymers such as polystyrene, polyethylene, polycarbonate, polyacrylamide, nylon or latex.

 The invention also relates to a cell genetically modified by transfection with an expression vector comprising the sequence of a DNA comprising a DNA encoding the 5-HT4D100A receptor.

 Preferably, the cell according to the invention is chosen from neuronal and / or epithelial cells.

 The invention also relates to a non-human animal model comprising cells genetically modified to express the 5-HT4D100A receptor.

Advantageously, said cells express the receptor constitutively.

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 The invention also relates to a method for identifying active compounds for treating or preventing a disease related to a failure in cAMP synthesis, of the type using a non-human animal model comprising cells genetically modified to express the receptor mutant 5HT4D100A receptor, wherein: a) a non-human mutant animal whose cells have been genetically engineered to express the mutated 5-HT4D100A receptor is administered with said chemical compound, under conditions permitting binding of said compound to the mutated receptor and b the action of said compound on cAMP synthesis is determined by any suitable means.

 The invention also relates to a method for the therapeutic treatment of a patient suffering from a pathology affecting cAMP synthesis, such as a neurodegenerative pathology, or a respiratory pathology, in particular asthma or cystic fibrosis comprising first administration to said patient, of the therapeutic composition of the invention, to induce overexpression of the mutated 5-HT4-D100A silent receptor in the tissues of said patient, in particular in the neuronal tissue for neurodegenerative pathologies or in the epithelial tissue for respiratory diseases.

 Once this overexpression has been obtained, these endogenous receptors are optionally stimulated by the synthetic agonists of said receptor.

Primperanm, le cisapride ou Prépulside# et le Tégazérode-HTF919 ou Zelmac) sont commercialisés ou Such 5-HT4 agonists (metoclopramide or

Primperanm, cisapride or Prepulside # and Tegazerode-HTF919 or Zelmac) are commercially available or

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 used clinically to increase gastrointestinal transit, without any central problems. The 5-HT4 agonists would also have a pro-mnemic effect, which is not necessarily a disadvantage in the case of a pathology of the elderly person.

 Advantageously, other synthetic ligands, which would be mutated receptor agonists and native receptor antagonists, could be used. Ligands having these properties would be particularly useful because these properties will limit the possible occurrence of undesirable central effects.

 Also, synthetic ligands that would be partial agonists of the native 5-HT4 receptor, and total agonists of the 5-HT4-D100A receptor could be used, during the activation phase of the receptor, to obtain an increase of the synthesis. cAMP.

 Surprisingly, the expression of the mutated receptor according to the technique disclosed is a relatively stable phenomenon that does not require many injections in the targeted regions under stereotactic control.

 It is possible, by using said receptors, to induce an increase in the intracellular concentration and cAMP synthesis that is completely controllable in intensity and in time, since they no longer respond to their endogenous ligand but only to specific exogenous ligands. .

 The Applicant has proved that such receptors, having a point mutation, for example 5-HT4A258L or

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 obtained by truncation of the C-terminal domain are constitutively active and have a natural ability to stimulate adenylate cyclase in the absence of any agonist (Claeysen et al Mol.Pharm 58: 136-144) (2000); Claeysen et al EMBO reports 21: 61-67 (2001).

 The invention will be better understood with the aid of the experimental work conducted by the Applicant in the context of the present invention as well as the appended figures in which: FIGS. 1A to 1D show that the agonists having a similarity of structure with serotonin are unable to stimulate the RASSL D100 receptor (3, 32) A.

 FIGS. 1A and 1B show the doseresponse curves for the wild-type and mutated D100 (3.32) A receptors exposed to tryptaminic derivatives (5MeOT, 5CT, HTF919 and 5-HT). CAMP production is measured in transiently expressing COS-7 cells, either the native 5-HT4a receptor or the mutated D100 (3.32) A receptor, at levels of 1500 230 and 1450 160 fmol / mg protein respectively.

 The production of cAMP is measured for 15 minutes and is expressed as a percentage of cAMP formation relative to control cells (cells transfected with the vector without the DNA encoding the receptor). In the control cells the conversion percentages of [3H] ATP to [3H] cAMP is 0.120.04%.

 The data represent the average values of four experiments performed by triplicate.

 Figure 1C illustrates the competitive binding assays performed for 5-HT in the presence of 0.24 nM [3H] GR113808.

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 The experiments were performed on membranes (8 g protein) extracted from COS-7 cells expressing similar levels of wild-type (WT) or mutated D100A receptors (3500 fmol / mg protein).

 FIG. 1D illustrates the Scatchard representation of the saturation experiments of the binding of the [3H] GR113808 compound (0.048 to 0.51 nM) on the 5-HT4a and 5-HT4D100 (3.32) A receptors expressed in COS cells. 7. RS 1000235 (10 M) was used to determine non-specific binding.

 FIGS. 2A and 2B show that the mutation of the conserved residue D100 (3.32) in alanine (A) does not prevent the response or the binding of a synthetic agonist of the 5-HT4-R receptor, the compound BIMU 8.

 FIG. 2A illustrates the effects of a range of concentrations of compound BIMU 8 on the intracellular levels of cAMP that were measured in COS-7 cells expressing the 5-HT4 (a) or D100A receptors at equivalent levels (FIG. 1350 60 and 1580 110 fmol / mg protein respectively).

 FIG. 2B illustrates the competition of compound BIMU 8 with compound [3H] GR113808. The conditions are identical to those used for the experiments of Figure 2A).

 Figures 3A and 3B show that the D100 (3,22) A mutation leads to changes in ligand properties, a wild-type antagonist becomes an agonist on the mutated receptor.

 FIGS. 3A and 3B show the activities of compounds ML10375 (A) and GR113808 (B), two potent and specific antagonists of the 5-HT4 (a) receptor. These two ligands were tested in COS-7 cells expressing

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 the wild-type 5-HT4 (a) receptor (2700 320 fmol / mg protein) or the mutated receptor (2500 280 fmol / mg protein).

 FIG. 3C shows the cAMP accumulation measured in COS-7 cells transiently expressing the wild-type and mutated D100 (3.32) A receptors at densities similar to those used in the absence (FIG. 3A) and in the presence (Figure 3B) 3x10-8 M BIMU 8.

 The ability of ML 10375 (10-5 M) to inhibit BIMU 8-induced cAMP accumulation was tested on the same COS-7 cells.

 FIG. 4 is a diagram showing the effects, in neurons, of cAMP induced after use of the silent receptor (RASSL) as a gene therapy tool, in particular for the treatment of Parkinson's disease.

EXPERIMENTAL WORK
1- Construction of the m5HT4a mutated receptor DNA.

 The mutant receptor was generated by exchanging the endogenous, aspartic acid (D) residue by the alanine (A) residue in the cDNA sequence of 5-HT-4 (a) R by site-directed mutagenesis through the use of a kit. Quick Change Site-Directed Mutagenesis of Stratagene.

 The sense primer used was: D100A - 5'-ACC TCT CTG GCT GTC CTA CTC ACC-3 '

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2- Cell culture and transfection
The cDNAs, subcloned into plasmid pRK5, were introduced into COS-7 cells by electroporation.

KZHP04 , 20 mM CH3Co2K, 2 0 mM KOH, 26,7 mM MgS04 ; pH 7,4) avec 25-2000 ng d'ADNc du récepteur identifié par la SEQ ID No. : 1 dans la liste de séquences en annexe. La quantité totale d'ADN a été maintenue constante à 15 g par transfection avec le vecteur pRK5 vide, comme entraîneur. Après 15 minutes à température ambiante, 300 l de la suspension cellulaire (107 cellules) ont été transférés à une cuvette d'électroporation de 0,4 cm (Bio-Rad, Ivry sur Seine, France) et les cellules ont reçu un choc électrique au moyen d'un appareil de pulsation de gènes (réglé à 1000 pF , 580 V). Les cellules ont été ensuite diluées dans du milieu d'Eagle modifié selon Dulbecco (DMEM, 106 cellules/ml) contenant 10% de sérum de veau f#tal décomplémenté et filtré (dSVF) et placées dans des boîtes de Pétri pour culture de cellules (15cm de diamètre) ou dans des plaques de 12 puits, à la densité souhaitée. The cells were trypsinized, centrifuged and resuspended in the next buffer (50 mM

KZHPO4, 20mM CH3Co2K, 20mM KOH, 26.7mM MgSO4; pH 7.4) with 25-2000 ng cDNA of the receptor identified by SEQ ID No. 1 in the attached sequence listing. The total amount of DNA was kept constant at 15 g by transfection with the empty pRK5 vector as a trainer. After 15 minutes at room temperature, 300 l of the cell suspension (107 cells) were transferred to a 0.4 cm electroporation cuvette (Bio-Rad, Ivry sur Seine, France) and the cells were shocked by means of a gene pulsation apparatus (set at 1000 pF, 580 V). The cells were then diluted in Dulbecco's modified Eagle's medium (DMEM, 106 cells / ml) containing 10% decomplemented and filtered fetal calf serum (dSVF) and placed in cell culture plates. (15cm in diameter) or in 12-well plates, at the desired density.

 3- Determination of cyclic AMP production (cAMP) in intact cells.

 After six hours of transfection, the medium in which the cells are found was replaced with dSVF-free DMEM containing tritiated adenine (2 μCi / ml) to label the ATP pool, and incubated overnight.

The level of cAMP formed was measured according to the method previously described (Dumuis A. Bouhelal R., Sebben M.,

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 Cory R. and Bockaert J. (1998) Mol. Pharmacol. 34: 880-887).

 4- Preparation of the membranes and binding assay of the radiolabelled ligand.

 The membranes were prepared from transiently transfected cells, plated in 15 cm diameter culture dishes, containing DMEM and 10% dSVF, for six hours and then incubated for 20 hours in patients. DMEM without dSVF. The cells were washed twice in PBS, scraped with a rubber spatula, put back into PBS and centrifuged at 4 ° C. (200 g for 4 minutes).

 The pellet was resuspended in buffer containing 10 mM HEPES (pH 7.4), 5 mM EGTA, 1 mM EDTA and 0.32 M sucrose and homogenized with a Teflon glass poter at 4 C. The homogenate was centrifuged at 20.000 g, 20 minutes and the membrane pellet was resuspended in 50 mM HEPES buffer (pH 7.4 at 5 mg / ml protein) and stored at -80 ° C until used.

 Saturation experiments were performed using a specific tritiated radiolabeled ligand, 3 [H] GR113808 at concentrations ranging from 0.048 nM to 1 nM.

 The densities of the 5-HT4 receptor were estimated with 3 [H] GR113808 at saturation concentrations (0.5 nM) according to previously described (Ansanay, H., Sebben, M., Bockaert, J. and Dumuis A. (1996) Eur.J.Pharmacol 298: 165-174).

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 Nonspecific binding was defined using either 5-HT (5x10-5 M) or RS 100235 (10-7 M). Protein concentrations were determined using a commercial protein quantitation assay (Bio-Rad).

Kd selon l'équation Kd=IC,0/1+S/KDs (Cheng,Y ., and Prussof,W.H. (1973) Biochem.Pharmacol.22 (23) ,3099-108) où S est la concentration de 3 [H] GR113808 et K la constante d' équilibre de 3 [H] GR113808 . 5- Data analysis
Competition and saturation experiments were analyzed by nonlinear regression calculations using the LIGAND computer program (Munson PJ, and Rodbard, D. (1980) Anal. Biochem. 107: 220-39). The saturation experiments were also analyzed according to the Scatchard representation. The IC50 values required for the displacement of 50% of the linkage of the, 3 [H] GR113808 have been converted into values

Kd according to the equation Kd = 1C, O / 1 + S / KDs (Cheng, Y., and Prussof, WH (1973) Biochem.Pharmacol.22 (23), 3099-108) where S is the concentration of 3 [ H] GR113808 and K the equilibrium constant of 3 [H] GR113808.

l'équation : Y= ( (Ymax-Ymin) /1 + (x/EC)1*) ) +ymin où EC50 (ou EC50inv) est la concentration de l'agoniste (ou de l'agoniste inverse), qui induit la moitié de la réponse maximale. ymax et ymin correspondent respectivement aux réponses maximales et minimales et nH est le coefficient de Hill. Les différences statistiques ont été examinées avec le programme Star-View Student (Abacus Concepts, Berkeley, Californie) avec les tests t. Using a Kaleidagraph program, the dose / response curves were calibrated according to

the equation: Y = ((Ymax-Ymin) / 1 + (x / EC) 1 *)) + ymin where EC50 (or EC50inv) is the concentration of the agonist (or inverse agonist), which induces half of the maximum response. ymax and ymin correspond respectively to the maximum and minimum responses and nH is the Hill coefficient. The statistical differences were examined with the Star-View Student program (Abacus Concepts, Berkeley, California) with the t tests.

<Desc / Clms Page number 24>

<Tb>
<tb> Compounds
<Tb>

GR 113308 1[2-méthylsulfonylamino)éthyll-4-

GR 113308 1 [2-methylsulfonylamino) ethyl-4-

<Tb>
<tb> piperidinyl <SEP> 1-methyl-indole-3-carboxylate
<tb> HTF <SEP> 919 <SEP> 5-methoxy-indole-3-carboxyaldehyde <SEP> 4pentyl-iminosemicarbazone
<tb> BIMU <SEP> Endo- <SEP> N- (8-methyl-8-azabicyclo [3.2.1] oct-
<tb> 3-yl) -2-oxo-3-isopropyl-2,3-dihydro-1H
<tb> Benzimidazole-1-carboxamise.
<Tb>

(S) -Zacopride <SEP> (S) -N- (1-SEP) azabicyclo [SEP] [2.2.2] oct-3-yl) -4-amino-5-cholo-2-methoxybenzamide
<tb> monohydrochloride
<Tb>

Cisapride Cis N- [1- [3- (4-fluorophenoxy)propyl] -3-

Cispride Cis N- [1- [3- (4-fluorophenoxy) propyl] -3-

<Tb>
<tb> methoxy-4-piperidinyl] -4-amino-5-chloro
<tb> 2-methoxybenzamide
<tb> Metoclopramide <SEP> N- (2-dimethylamino) ethyl) -4-amino-5-chloro-2-methoxy-benzamide
<tb> Renzapride <SEP> () <SEP> endo-N- (1-azabicyclo [SEP] [3.3.1] <SEP> non-4yl) -4-amino-5-chloro-2-methoxy-benzamide
<tb> monohydrochloride
<tb> SB <SEP> 204070 <SEP> (1-butyl-4-piperidinyl) methyl <SEP> 8-amino-7-chloro-1,4-benzodioxane-5-carboxylate
<Tb>

ML 10302 2-(l-pipéridinyl)éthyl 4-amino-5-chloro-

ML 10302 2- (1-piperidinyl) ethyl 4-amino-5-chloro

<Tb>
<tb> 2-methoxybenzoate
<tb> ML <SEP> 10735 <SEP> 2- (cis-3,5-dimethyl-1-piperidinyl) ethyl4amino-5-chloro-2-methoxybenzoate
<tb> RS <SEP> 67333 <SEP> 1- (4-amino-5-chloro-2-methoxy-phenyl) -3-
<Tb>

(1-butyl-4-pipéridinyl)-1-propanone RS 100350 N-[2-[4-[3-(8-amino-7-chloro-l,4-

(1-Butyl-4-piperidinyl) -1-propanone RS 100350 N- [2- [4- [3- (8-amino-7-chloro-1,4)

<Tb>
benzodioxan-5-yl) -3-oxo-propyl] -piperidinyl] ethyl] -4-methoxybenzenesulfonamide
<tb> RS <SEP> 124523 <SEP> 1- (4-amino-5-chloro-2-methoxy-phenyl) -3-
<Tb>

[1- [3-4-fluorophényl)propyl] -4-

[1- [3-4-fluorophenyl) propyl] -4-

<Tb>
<tb> piperidinyl] -1-propanone
<tb> RS <SEP> 47431 <SEP> 1- (4-amino-5-chloro-2-methoxy-phenyl) 3-
<Tb>

[1-[3-(3,4-diméthoxyphényl)propyll-4-

[1- [3- (3,4-dimethoxyphenyl) propyll-4-

<Tb>
<tb> piperidinyl] -1-propanone
<tb> RS <SEP> 100235 <SEP> 1- (8-Amino-7-chloro-1,4-benzodioxan-5-yl) <SEP> -3- <SEP> [1- <SEP> [3-3, 4-dimethoxyphenyl) propyl] <SEP> - <SEP>
<Tb>

4-pipéridinyl]-1-propanone
RESULTATS ET DISCUSSION
Les résultats ci-dessus illustrent pour la première fois l'activation d'un récepteur 5-HT4, connu

4-piperidinyl] -1-propanone
RESULTS AND DISCUSSION
The above results illustrate for the first time the activation of a known 5-HT4 receptor

<Desc / Clms Page number 25>

 for its insensitivity to its endogenous ligand (Mialet J. et al British Journal of Pharmacol (2000) 130: 527-538), by synthetic non-primary amine agonists.

 Based on Strader's work on (3-adrenergic and other biogenic amine receptors (Straders, C., Fong, TT Tota, M., Underwood, D. and Dixon, R. (1994) Annu .Rev.Biochem.

63, 101-132), it has been observed that the key interaction between these receptors and the ligand is at the level of the TM III aspartic acid residue, highly conserved throughout the family of biogenic amine receptors.

l'aspartate, en position 100 dans le TM-III (Dloo (3 . 32 ) . In the case of the 5-HT4 receptor it is

aspartate, at position 100 in the TM-III (Dloo (3, 32).

This residue appears to intervene in an ionic interaction between its negatively charged carboxylate group and the positively charged nitrogen of the protonated amine of the neurotransmitter (5-HT and tryptamine derivatives).

 The Applicant has now proved that the mutation of D100 (3.32) in alanine in the 5-HT4 receptor which is not stimulable by 5-HT is also not by other tryptamines whose structure is very close to that of the 5 -HT (5-CT or 5-MeOT) or by a substituted indolated carbazimidamide derivative (HTF-919) (Buchheit KH, Gamse, R, Giger, R, Hoyer, D, Klein, F, Kloppner, E., Pfannkuche, HJ and Mattes HJMed.Chem (1995) 38 (13): 2326-30), (Figures 1A, 1B).

 Mutations of a wide range of other, highly conserved residues in the family of biogenic amine receptors involved in the

<Desc / Clms Page number 26>

 5-HT (S197 (5.43), W275 (6.48) and F275 (6.51)) were also performed. However, none of these mutations led to completely removing the responses of both 5-HT and other tryptamine derivatives. The mutated F275A receptors, W2722A and S197A, have only a reduced affinity of 50 to 5000-fold for 5-HT and its derivatives, with no change in the stimulation maximum (data not shown).

Dloo (3 .32) A, (Figure 1D) . Conversely, the mutation of the highly conserved D100 residue (3.32) in the 5-HT4 receptor only very slightly affects the binding of the radiolabelled specific ligand 3 [H] GR113808 when this amino acid is mutated to alanine ( Kd = 0.17 0.008 nM and 0.38 0.09 nM respectively, in the native and mutated receptor

Dloo (3 .32) A, (Figure 1D).

 Using the ligand 3 [H] GR113808, the Applicant verified that the 5-HT ligand is unable to bind to the mutated D100 (3.32) A receptor (Figure 1C).

 The binding of 3 [H] GR113808 maintained on the mutated receptor was the first observation which indicated that the presence of a basic nitrogen in the aromatic ring of the side chain, associated with an increase in the distance between said basic nitrogen and the The aromatic ring removed the need for the presence of the carboxylic group of D100 (3.32) for binding with its ligand.

 This was confirmed when the Applicant screened most nontryptaminic 5-HT4 receptor agonists for their ability to activate the 5-HT4D100A receptor.

 The compound BIMU 8, belonging to the class of azabicycloalyl, has been demonstrated

<Desc / Clms Page number 27>

benzymidazolones (Dumuis A, Sebben M, Monferini E, Nicola M, Turconi M, Ladinsky H, Bockaert J. Naunyn Schmiedebergs Arch Pharmacol (1991) 343: 245-51) very structurally different from 5-HT, is a very good 5-HT4 receptor agonist
As shown in Figure 2, this compound remains fully active, with the same efficiency, on the mutant D100A receptor as on the wild-type 5-HT4 receptor.

EC50 values for stimulation of cAMP were 4 1.5 nM and 1 0.5 nM, respectively, for the wild-type and mutated receptor (Figure 2A).

 The affinity of BIMU compound 8 for the 5HT4 receptor is also better on the D100A mutant receptor than on the wild-type receptor. The Kd values for compound BIMU 8, measured by competition with radiolabelled ligand 3 [H] GR 113808, are, respectively, 11 nM and 6, 3 nM for the wild-type receptor and the mutant (D100A) (FIG. 2B). ).

 In a similar manner, the benzamides bearing the 2-methoxy-4-amino-5-chloro substitution, such as the compounds Renzapride, S-zacopride or cisapride, are equivalent in terms of their efficacy on the wild-type receptor and on the mutated receptor (D100 (3.32) A (Table I) with nanomolar affinities, only a small decrease in their affinity for the mutated D100A receptor is observed.

récepteur 5-HT4 muté Dloo (3 , 32 ) A. Table I below shows the efficacy of 5-HT4 receptor ligands on the wild-type receptor and the

5-HT4 mutated Dloo receptor (3, 32) A.

<Desc / Clms Page number 28>

TABLE I

<Tb>
<tb> Drugs <SEP> 5-HT4 (a) <SEP> 5-HT4-RD100A
<tb> (M) <SEP>% <SEP> Answer <SEP>% <SEP> of <SEP> The <SEP> Answer
<tb> 5-HT <SEP> BIMU8
<Tb>

.. BIMU8 o"T f 5-HT 100 ""on.-. "ï Bimu 8 90 10 100 :n:ifC5:\:';;::>:': rir≥o i1;;:}:i>:':;:;:,,:t,r:,:;::'::;;rca:, ,5-met ::t: H'rF9 imJh 5-Meot 90 10 HTF919 eJ >:::;;;<;0;:;:,', HTF 919 66 15 Metoclopramide 20 10 20 10

## EQU1 ## where: ## EQU1 ## where: ## EQU1 ## ## EQU00006 ##, ## STR2 ## : Metoclopramide 20 10 10

<Tb>
<tb> Cisapride <SEP># N # O # <SEP> Renzapride <SEP> 85 <SEP> 8 <SEP> 100 <SEP>
<tb> @ <SEP>## NH # <SEP> S-Zacopride <SEP> 100 <SEP> 5 <SEP> 100 <SEP> 5
<tb> H2N # OMe <SEP> Cisapride <SEP> 100 <SEP> 10 <SEP> 100 <SEP> 10
<tb> ML <SEP> 10302 <SEP> 57 <SEP> 9 <SEP> 100 <SEP> 6 <SEP>
<tb> SB <SEP> 204070 <SEP> 20 <SEP> 3 <SEP> 100 <SEP> 5
<tb> RS <SEP> 100350 <SEP> 17 <SEP> 5 <SEP> 70 <SEP> 10
<Tb>

RS67333 -a>fV RS 124523 21 6 75 5

RS67333 -a> fV RS 124523 21 6 75 5

<Tb>
<tb> H2N # OMe <SEP> N-Bu-n <SEP> RS <SEP> 67333 <SEP> 28 <SEP> 6 <SEP> 85 <SEP> 12
<tb> RS <SEP> 47431 <SEP> 30 <SEP> ~ <SEP> 5 <SEP> 100 <SEP> ~ <SEP> 7
<Tb>

V ",,/,'/,',,,,',',',"///;>0'; GR 113808 10 9 78 10

V ",, /, '/,' ,,,, ',', ',"//;;>0'; GR 113808 10 9 78 10

<Tb>
<tb> GR113808 <SEP>## N
<tb> ML <SEP> 10375 <SEP> Cl # O # N # <SEP> ML <SEP> 10375 <SEP> 5 <SEP> ~ <SEP> 4 <SEP> 80 <SEP> 15 <SEP>
<Tb>

Eï::;[@iht:ft!1Jjj: :t::t:

EI ::; [@ iht: ft! 1Jjj:: t :: t:

<Desc / Clms Page number 29>

Pour le récepteur muté 5-HT4-D1 (3, 32)A, les résultats sont exprimés en pourcentage de l'accumulation d'AMPc induit par le composé BIMU8. Efficacy measurements were determined in cAMP accumulation experiments in transiently expressing COS-7 cells, either the 5-HT4a receptors or the mutated D100A receptor at the same receptor density (about 2500fmol / mg of protein). For the wild-type receptor the results are expressed as a percentage of the 5-HT induced cAMP accumulation.

For the mutated 5-HT4-D1 (3, 32) A receptor, the results are expressed as a percentage of the BIMU8 compound-induced cAMP accumulation.

 These data suggest that the mutated D100 (3.32) A receptor can be as activated as the wild-type receptor if the compound can bind to the receptor. Expression levels of wild and mutated receptors are not significantly different. In contrast, the mutant W272 (6.51) A receptor had an abnormally low level of expression. The maximum level of protein expression was 600 fmol / mg protein regardless of the amount of transfected DNA (Roth B. L., Shoham, M. Choudhary, M.S., Khan, N.

Mol.Pharmacol (1997) 52: 259-66).

 Two drugs are antagonists of the wild-type receptor, whereas they are mutated receptor agonists (D100 (3.32) A).

 The benzoate derivatives bearing the 2-methoxy-4-amino-5-chloro substitution (such as ML10302) (Langlois, M., Zhang, L., Yang, D., Bremont, B., Shen, S., Manara, L. and Croci, T. (1994) Bioorg.Med.Chem.Lett.4: 1433-1436) and the corresponding aryl ketones (such as RS67333, RS124523, RS1000350, RS47431) (Clark, RD (1998) In 5 -HT4 receptors in the brain and periphery.

(Eglen, R.M., ed pp.1-48, Springer), are partial agonists of 5-HT4 receptors.

<Desc / Clms Page number 30>

 As shown in Table I, they are more effective on the mutated D100 (3.32) A receptors than on the wild-type receptor.

 Most interestingly, another benzoate derivative, known to be a highly specific 5-HT4 ligand antagonist (ML 10375) (Yang, D, Soulier, JL., Sicsic, S., Mathe-Allainmat, M., Bremont, B., Croci, T., Cardamone, R., Aureggi, G. and Langlois, MJMed.Chem (1997) 40: 608-21) or a 5-HT4 receptor inverse agonist (Blondel, O.). , Gastineau, M., Langlois, M., Fischmeister, R. Br.J.Pharmacol. (1998) 125: 595-7.19), proves to be a total agonist of the mutated 5-HT4 receptor D100 (3.32). A (ECso = 1.06 nM), (Figures 3A and 3C).

 These results led the Applicant to screen the properties of putative agonists of a series of 5-HT4 receptor antagonists. The most commonly used radiolabelled antagonist 3 [H] GR 113808 has been found to be the most commonly used radiolabelled antagonist (Figure 3C) for 5-HT4 receptor studies (Grossman, CJ, Kilpatrick, GJ, Bunce, KT Br .J.Pharmacol.

(1993) 109: 618-24) is a potent and effective agonist (EC50 = 0.17 0.04 nM) against the mutated D100 (3.32) A receptor (Figure 3B).

The above results show that the mutated 5-HT4 receptor D100 (3.32) A is a RASSL-type receptor, unique on the basis of several characteristics:
1) It is generated by a single mutation.

 2) It is completely insensitive to its unique endogenous natural agonist: 5-HT.

 3) It can be stimulated by a long series of synthetic agonists of different chemical classes.

<Desc / Clms Page number 31>

 4) Many of these synthetic ligands have nanomolar affinity for RASSL 5-HT4 and some of them are available for oral administration, such as Cisapride.

 Cisapride or PrepulsideTM has been used for many years for the treatment of dyspepsia and mainly gastro-oesophageal reflux in children. This compound was withdrawn from the market because of some arrhythmias caused by activation of potassium channels (K +) (Kii, Y., Ito, T.

J.Cardiovasc.Pharmacol. (1997) 29: 670-5). Other benzamides are still in development and will certainly be available for administration to humans.

 Interestingly, RS 67333 has been shown to be another potent agonist of RASSL 5-HT4. This compound also has a great ability to cross the blood-brain barrier.

 5) Compounds (ML 10375 and GR 113808) are highly specific antagonists of the native receptor and become agonists of the RASSL 5-HT4 receptor. The compound GR 113808 also crosses the blood-brain barrier which makes it particularly interesting for putative use of the receptor in vivo.

 Thus, with these compounds (ML 10375 and GR 113808), it is possible to keep the native receptors silent while activating the RASSL 5-HT4 receptor, including when expressed by genetic engineering in mice.

 Compared with beta-adrenergic RASSL receptors coupled to G proteins

<Desc / Clms Page number 32>

 previously described, the RASSL 5-HT4 receptor has the advantage of being stimulated by synthetic compounds of a much higher affinity: nM for the RASSL 5-HT4 receptor whereas the affinity is only M for the receptor Beta2-adrenergic RASSL (Small KM, Brown KM, Forbes SL, Liggett SB, J. Biol Chem (2001) 276 (34): 31596-601).

 Structural analyzes of 5-HT4 receptor ligands, used in this study, are consistent with a recent report on comparative mapping of 5-HT4 receptor binding sites (Lopez-Rodriguez, ML, Murcia, M., Benhamu, B., Viso, A., Campillo, M. and Pardo, L. Bioorg.Med.Chem.Lett (2001) 11 (21): 2807-11.22).

 The structural features that define the 5-HT4 receptor ligands are an aromatic moiety, a coplanar carbonyl, a carboxylic or ketone moiety, and a basic nitrogen atom.

 The basic nitrogen substituent must be bulky (as in all active drugs for the described RASSL 5-HT4 receptor) in order to stabilize in a hydrophobic pocket of the receptor, without requiring the presence of the carboxylate group of aspartate D100.

 The size of the substituent, to some degree, improves the selectivity and affinity of the ligand for its receptor.

 One of the assumption of the applicant is that the basic nitrogen of these drugs interacts with said hydrophobic pocket with a negative charge, but not with the aspartate D1003,32.

 This Asp residue is, on the other hand, necessary for binding with the protonated amine present in the derivatives

<Desc / Clms Page number 33>

 tryptamines close to 5-HT. These compounds are therefore inactive on the RASSL 5-HT4 receptor described.

It can therefore be assumed that the interaction of basic nitrogen is not essential. The affinity of ligands with bulky substituents for the RASSL 5-HT4 receptor (Structures in Table 1) will depend on the interactions between their hydrophobic cycle and the hydrophobic pocket defined by the highly conserved aromatic residues of TMVI and VII (W6,48, F6.51 and Y7.43) (16, 23, 24) .s
This RASSL 5-HT4 receptor therefore constitutes a very useful model for determining the physiological effects of the expression of a Gs-protein coupled receptor and for controlling, for example in transgenic mouse models, the signal transduction linked to Gs proteins. in vivo (Scearce-Levie, K., Coward, P., Redfern, CH, Conklin, BR Methods Enzymol.

(2002) 232-48.

 When said receptor is expressed, it is functionally silent until the time of administration of the synthetic ligand.

 Such a receptor is particularly useful for regulating numerous physiological responses that require activation of the Gs-coupled pathway.

 By way of example, it is possible to propose the expression of a RASSL 5-HT4 receptor in the heart of a mouse, which does not express a 5-HT4 receptor, unlike the human heart (Kaumann, AJ, Lynham, JA, Brown, AM

Naunyn. Schmiedebergs. Arch. Pharmacol. (1996) 353 (5): 592-5). This model would test the chronic or acute activation of the Gs path, in

<Desc / Clms Page number 34>

 no stimulation of other peripheral targets, including vascular tissue.

 At the central level, the RASSL 5-HT4 receptor, expressed in the substantia nigra can be stimulated and the production of cAMP in dopaminergic neurons will lead to their survival and protection when they are treated by a neurotoxic drug, the MPP + ( 1-methyl-4-phenylpyridinium) the active metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine).

 It is known that the production of cAMP in vivo, allows such protection. Such experiments provide a basis for gene therapy for Parkinson's disease.

Claims (10)

 HT4-DlooA, in which the codon encoding aspartate10o, of the transmembrane domain III of the corresponding wild-type receptor, is mutated to encode an alanine, for the preparation of a medicament for the treatment and / or prevention of a pathology caused by dysfunction in cAMP synthesis.

 1) Use of a cDNA encoding a receptor  2) Use according to claim 1, characterized in that the sequence of the cDNA of the 5-HT4-D100A mutated is the sequence identified under the number SEQ ID No. 1 in the attached sequence listing.  3) The use according to claims 1 or 2, characterized in that said pathology caused by malfunction in the synthesis of cAMP is a neurodegenerative disease, preferably Parkinson's disease, Alzheimer's disease or Hungtington's Chorea.  4) Use according to claims 1 or 2, characterized in that said pathology caused by a malfunction in the synthesis of cAMP is a respiratory disease, preferably asthma or cystic fibrosis.  5) Use according to claims 1 to 4, characterized in that said nucleic acid sequence is inserted into a viral or nonviral transfer vector.  6) Use according to claim 5, characterized in that the viral vector is adapted for the transfer of the nucleotide sequence in a cell, and that it comprises the regulatory elements necessary for the expression of said receptor to allow targeted expression and functional of it. <Desc / Clms Page number 36>  7) Use according to claim 6, characterized in that the transfer vector is a viral vector selected from the group of vectors comprising vectors derived from viruses associated with adenovirus type 2, vectors from the lentivirus with different pseudotypes, vectors from feline immunodeficiency viruses and vectors derived from Foamy virus.  8) The use according to claim 5, characterized in that said transfer vector is a plasmid comprising the complete genome of the adenovirus-associated vector (AAV) (psub201) and the packaging plasmid (pAAV / Ad).  9) The use according to claim 8, characterized in that said vector is the PAAV-vector.

 promoter-5-HT4-D 100 A-HA constructed in a cassette containing a strong promoter.  10) The use according to claims 6 to 9, characterized in that said vector comprises a promoter, selected from a group comprising the actin promoter / cytomegalovirus enhancer (or CAG promoter) and the dopamine transporter promoter.