Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-12-28
2001-08-14
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S022000, C530S300000, C530S350000, C514S012200
Reexamination Certificate
active
06274333
ABSTRACT:
The present invention relates to a polypeptide which constitutes a subtype of the neurotensin receptor, known as the type-2 neurotensin receptor (NT-R2), in mammals.
Neurotensin (NT) is a tridecapeptide involved in intracellular communication. It acts as a hormone in peripheral organs and as a neurotransmitter in the central nervous system (Carraway R. et al., Peptides, 1982, 3, 115-123 and Maj. J. K. et al., Neuroscience, 1987, 22, 499-524). In particular, it modulates the transmission of dopamine in the nigrostriatal and mesolimbic pathways (Nemeroff, C. B., Psychoneuro-endocrinology, 1986, 11, 15-37 and Kitabgi, P., Neurochem. Int., 1989, 14, 111-119). In addition, NT plays a role in nociception, in hypothermia, in controlling the secretion of hypophyseal hormones and in muscle relaxation (Vincent, J. P., Cell. Mol. Neurobiol., 1995, 15, 501-512).
In adult rat brain, neurotensin binds at two distinct sites, which are distinguishable, on the one hand, by their sensitivity to levocabastine, an antagonist of the histamine H1 receptor, and, on the other hand, by their differential affinity for neurotensin (Schotte A. et al., Naunynschmiedeberg's Arch. Pharmacol., 1986, 333, 400-405). The low-affinity levocabastine-sensitive site appears after birth and has a distribution in the brain which is different from that of the high-affinity levocabastine-insensitive site (Schotte A. et al.,
Brain. Res.,
1987, 408, 326-328).
The high-affinity levocabastine-insensitive receptor has been cloned from rat (Tanaka K. et al., Neuron, 1990, 4, 847-854) and from man (Vita N. et al., FEBS lett., 1993, 317, 139-142 and Watson M. et al., Mayo Clin. Proc., 1993, 68, 1043-1048). This receptor represents less than 30% of the NT binding sites in the adult rat brain; it has been found mainly in substantia nigra and in the ventral tegmental area. This cloned rat neurotensin receptor is a 424-amino-acid protein which belongs to the G-protein-coupled superfamily of receptors. The biochemical and pharmacological properties of this receptor have been studied extensively. It has been demonstrated that, upon activation, this receptor modulates the intracellular amount of cGMP (Amar S. et al., Biochem. Biophys. Res. Commun, 1985, 129, 117-125), of cAMP (Bozou J. C. et al., Biochem. J., 1989, 264, 871-878 and Yamada M. et al., Eur. J. Pharmacol., 1993, 244, 99-101) and of inositol phosphates (Watson M. A. et al., J; Neurochem., 1992, 59, 1967-1970 and Hermans E. et al., Mol. Pharmacol., 1996, 49, 365-372).
The recently discovered non-peptide NT antagonist, {2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-di-methoxyphenyl)pyrazol-3-yl)carbonylamino]tricyclo-(3.3.1.1.3.7)decane-2-carboxylic} acid known under its code name SR 48692 and described in EP 477,049, inhibits the binding of NT to the cloned receptor (Gully D et al, Proc. Natl. Acad. Sci. USA, 1993, 90, 65-69) However, a recent study shows that in mice and in rats, the hypothermia and analgesia induced by NT are insensitive to SR 48692 (Dubuc I. et al., Br. J. Pharmacol., 1994, 112, 352-354), therefore suggesting that NT might act differently through different receptor subtypes.
It has also been demonstrated that SR 48692 may affect differently the NT-induced behaviour and changes in dopaminergic transmission (Poncelet M. et al., Naunynschmiedeberg's Arch. Pharmacol., 1994, 349, 57-60 and Steinberg R. et al., Neuroscience, 1994, 59, 921-929). Besides pharmacological arguments in favour of NT receptor subtypes, a recent study comparing the distributions of the neurotensin receptor and the transcripts of the cloned neurotensin receptor in rat brain has suggested the existence of another neurotensin receptor (Nicot A. et al., J. Comp. Neurol. 1994, 59, 921-929 and Le F. et al., Trends Pharmacol. Sci., 1996, 17, 1-3). The neurotensin receptor transcripts are detected in different regions of the brain, but not in the subfornical region, although the neurotensin binding sites have been clearly associated with the cell bodies of this region (Nicot A. et al., J. Comp. Neurol., 1994, 59, 921-929).
Moreover, a novel and very powerful neurotensin antagonist has recently been described (Gully et al.): 2-{(5-(2,6-dimethoxyphenyl)-1-(4-(N-(3-dimethylamino-propyl)-N-methylcarbamoyl)-2-isopropylphenyl)-1H-pyrazole-3-carbonyl)amino}adamantane-2-carboxylic acid hydrochloride, known under the code name SR 142948 A. This compound has an affinity of the order of a nanomole and completely displaces [
125
I]-neurotensin and [
3
H]-SR 48692 from their binding with the type-1 receptor (Gully et al.).
SR 142948 A antagonizes the effects in vitro (formation of IP1 in HT29 cells (IC
50
=3.9 nM) or mobilization of intracellular calcium ions in CHO cells transfected with NT-1R) and in vivo of neurotensin (release of acetylcholine in rat striatum, hypothermia and analgesia in rats and mice, rotation in mice).
The inventors have demonstrated the existence of a novel neurotensin receptor subtype in mammals, known as the NT-R2 receptor. More particularly, they have managed to clone and sequence the NT-R2 receptor of rats (rNT-R2), (Chalon P., et al., FEBS Letters, 1996, 386, 91-94), and the human NT-R2 receptor (hNT-R2).
The inventors have also been able to study the pharmacological profile of the NT-R2 receptor: the NT-R2 receptor has a strong affinity for neurotensin as well as for xenine, neuromedin and levocabastine, is insensitive to the compound SR 48692 and binds the compound SR 142948 A strongly.
The NT-R2 receptor is a membrane protein consisting of seven transmembrane domains, preceded by a long extracellular amino-terminal domain and followed by a long intracellular carboxy-terminal domain. Hydropathic analysis of the NT-R2 according to the invention indicates eight hydrophobic regions of approximately 20 amino acids, corresponding to a potential signal peptide at the NH
2
end, plus seven transmembrane domains. After removal of the signal peptide, the receptor representing the invention has a molecular weight of approximately 40-45 kilodaltons.
The NT-R2 receptor is coupled in vivo to G proteins which allow transduction of the signal via the phospholipase C pathway.
The present invention thus relates to an isolated polypeptide which constitutes the type-2 neurotensin receptor (NT-R2).
More particularly, the invention relates to a purified polypeptide which constitutes the type-2 neurotensin receptor of rats (rNT-R2) and of man (hNT-R2).
More particularly, the subject of the invention is an hNT-R2 polypeptide comprising the amino acid sequence SEQ ID No. 2 or the amino acid sequence SEQ ID No. 4, or any polypeptide fragment or derivative thereof which is biologically active.
The sequence SEQ ID No. 2 represents the amino acid sequence of the hNT-R2 polypeptide. The seven potential transmembrane domains of the protein are underlined.
The sequence SEQ ID No. 4 represents the amino acid sequence of the rNT-R2 polypeptide.
The term “derivative” is understood to refer to any polypeptide variant of the polypeptide having the sequence SEQ ID No. 2 or No. 4 or any molecule resulting from a genetic and/or chemical modification of the sequence SEQ ID No. 2 or No. 4, that is to say obtained by mutation, deletion, addition, substitution and/or chemical modification of a single or of a limited number of amino acids, as well as any isoform sequence, that is to say a sequence which is identical to the sequence SEQ ID No. 2 or No. 4, to one of its modified sequences or fragments, containing one or more amino acids in the form of the D enantiomer, the said iso-form, modified or variant sequences having conserved at least one of the properties which makes them biologically active.
The expression “biologically active” means that the compound to which it refers is capable of binding to neurotensin or to ligands related to neurotensin and/or of participating in the transduction of the signal induced by neurotensin in the cell membrane, and/or capable of inducing antibodies which recognize the NT-R2 polypeptide according to the invention. Th
Caput Daniel
Chalon Pascale
Ferrara Pascual
Vita Natalio
Jacobson & Holman PLLC
Leary Louise N.
Sanofi-Synthelabo
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