Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-08-15
2003-01-21
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S069100, C435S070100, C435S471000
Reexamination Certificate
active
06509163
ABSTRACT:
TECHNICAL FIELD
The present invention relates, in general, to a purinergic receptor and, in particular, to a P2X
7
(also designated P2Z) receptor. The invention further relates to a nucleic acid encoding the P2X
7
receptor and to a method of producing P2X
7
recombinantly using same. The invention also relates to a method of screening compounds for their ability to inhibit P2X
7
activity and thereby for their usefulness in treating a variety of diseases/disorders, including arthritic and respiratory disorders and neurodegenerative diseases.
BACKGROUND
Cell surface receptors for ATP can be divided into metabotropic (P2Y/P2U) and ionotropic (P2X) classes. The metabotropic class belongs to the superfamily of G protein-coupled receptors, with seven transmembrane segments. The ionotropic class members (P2X
1
-P2X
6
) are ligand-gated ion channels, currently thought to be multisubunit proteins with two transmembrane domains per subunit (Buell et al, Europ. J. Neurosci. 8:2221 (1996)). P2Z receptors have been distinguished from other P2 receptors in three main ways (Buisman et al, Proc. Natl. Acad. Sci. USA 85:7988 (1988); Cockcroft et al, Nature 279:541 (1979); Steinberg et al, J. Biol. Chem. 262:3118 (1987)). First, activation of P2Z receptors leads not only to an inward ionic current, but also to cell permeabilization. Second, 3′-O-(4-benzoyl)benzoyl ATP (BZATP) is the most effective agonist, and ATP itself is of rather low potency. Third, responses are strongly inhibited by extracellular magnesium ions, which has been interpreted to indicate that ATP
4−
is the active agonist (DiVirgilio, Immunol. Today 16:524 (1995)).
A seventh member of the P2X receptor family has been isolated from a rat cDNA library and, when expressed in human embryonic kidney (HEK293) cells, exhibits the above three properties (Surprenant et al, Science 272:735 (1996)). This receptor (rP2X
7
) thus corresponds to the P2Z receptor. rP2X
7
is structurally related to other members of the P2X family but it has a longer cytoplasmic C-terminus domain (there is 35-40% amino acid identity in the region of homology, but the C-terminus is 239 amino acids long in the rP2X
7
receptor compared with 27-20 amino acids in the others). The rP2X
7
receptor functions both as a channel permeable to small cations and as a cytolytic pore. Brief applications of ATP (1-2s) transiently open the channel, as is the case of other P2X receptors. Repeated or prolonged applications of agonist cause cell permeabilization (which permeabilization involves the cytoplasm C-terminus); reducing the extracellular magnesium concentration potentiates this effect.
The P2Z receptor has been implicated in lysis of antigen-presenting cells by cytotoxic T lymphocytes, in the mitogenic stimulation of human T lymphocytes, as well as in the formation of multinucleated giant cells (Blanchard et al, Blood 85:3173 (1995); Falzoni et al, J. Clin. Invest. 95:1207 (1995); Baricolrdi et al, Blood 87:682 (1996)). However, the interpretation of the physiological role of P2X
7
receptor has been complicated by functional differences which seem to exist between rodent and man (Hickman et al, Blood 84:2452 (1994)). The human macrophage P2X
7
receptor (hP2X
7
) has now been cloned and its functional properties determined (Rassendren et al, J. Biol. Chem. 272:5482 (1997)).
SUMMARY OF THE INVENTION
The present invention relates to a P2X
7
receptor and to a nucleic acid encoding same. The invention also relates to a method of producing P2X
7
recombinantly and to a method of screening compounds for their ability to inhibit P2X
7
activity. Compounds selected using the present screen can be used to treat a variety of physiological diseases and disorders.
Objects and advantages of the present invention will be clear from the description that follows.
REFERENCES:
patent: 5360893 (1994-11-01), Owens et al.
patent: WO 95/33048 (1995-12-01), None
Coutinho-Silva et al. , Characterization of p2z purinergic receptors on phagocytic cells of the thymic reticulum in culture (Apr. 26, 1996), Biochem. et Biophys. Acta, 1280: 217-218.*
Wiley JS et al. , The P2z-purinoceptor of human lymphocytes: actions of nucleotide agonists and irreversible inhibition by oxidized ATP (1994) Br. J. Pharmacol. 112: 947-950.*
Suprenant A, The cytosolic P2z receptor for endogenous ATP identified as a P2X receptor (P2X7). (May 3, 1996), Science 272: 735-737.*
Rassendren F, The permeabilizing ATP receptor, P2X7 ( Feb. 28, 1997) J of Biol. Chem. 28: 5482-86.*
Molecular Probes Catalogue Supl. Cell Biology and Imaging. p5. Molecular Probes, Inc. Eugene, OR. 2001.*
Bo et al., “Solubilization and Molecular Size Determination of the P2xPurinoceptor from Rat Vas Deferens*”, The Journal of Biological Chemistry, vol. 267, No. 25, Sep. 5, 1992, pp. 17581-17587.
Buell et al., “Mini-Review P2X Receptors: An Emerging Channel Family”, European Journal of Neuroscience, vol. 8, 1996, pp. 2221-2228.
Owens et al., “Identification of mRNAs Associated with Programmed Cell Death in Immature Thymocytes”, Molecular and Cellular Biology, vol. 11, No. 8, Aug. 1991, pp. 4177-4188.
Simon et al., “Solubilisation and Molecular Characterisation of the P2xPurinoceptor”, Biochemical Society Transactions, vol. 21, No. 2, May 1993, p. 200S.
Surprenant et al., “The Cytolytic P2ZReceptor for Extracellular ATP Identified as a P2XReceptor (P2X7)”, Science, vol. 272, May 3, 1996, pp. 735-738.
Valera et al., “A New Class of Ligand-Gated Ion Channel Defined by P2XReceptor for Extracellular ATP”, Nature, vol. 371, Oct. 6, 1994, pp. 516-519.
GenBank Accession No. Y09561, H. sapiens mRNA for P2X7 receptor, accessed Dec. 20, 1998.
Valera et al., “Characterization and chromosomal localization of a human P2X receptor from urinary bladder”, Receptors and Channels 3(4):283-289 (1995).
An ATP-gated cation channel with some P2Z-like characteristics in gastric smooth muscle cells of toad, J. Physiol. 498(Pt. 2):427-442 (1997).
Rassendren et al, “The premeabilizing ATP receptor, P2X7”, J. Biol. Chem. 272(9):5482-5486 (1997).
Murgia et al, “Oxized ATP”, J. Biol. Chem. 268(11):8199-8293 (1997).
Buell et al, “P2X receotprs: an emerging channel family”, Eur. J. Neurosci. 8(10):2221-2228 (1996).
GenBank Accession No. X95882, R. novegicus mRNA for ATP ligand gate ion channel, accessed Dec. 20, 1998.
Humphreys et al, Induction of the P2Z/P2X7 nucleotide receptor and associated phospholopase D activity by lipopolysaccharide adn INF-gamma in the human THP-1 monocytic cell line, J. Immunol. 157(12):5627-5637 (1996).
Di Virgilio et al, “A purinergic hypothosis for immunomoldulation”, Ital. J. Biochem. 45(4):195-203 (1996).
Dubyak et al, “Expression of multiple ATP receptor subtypes during the differentiation and inflammatory activation of myeloid leukocytes”, Drug Dev. Res. 39:269-278 (1996).
Wiley et al, “P22z-purinoceptor of human lymphocytes: actions of nucleotide agonists and irreversible inhibition by oxidized ATP”, Br. J. Pharmacol. 112:946-950 (1994).
Buell Gary Nutter
Kawashima Eric
Surprenant Annmarie
Glaxo Group Limited
Kunz Gary L.
Landsman Robert S.
Nixon & Vanderhye P.C.
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