Method of screening for compounds that bind P2x receptor

Details Method of screening for compounds that bind P2x receptor Method of screening for compounds that bind P2x receptor

1999-07-30

2001-02-27

Mertz, Prema (Department: 1646)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S069100, C514S002600, C514S012200, C530S350000

Reexamination Certificate

active

06194162

ABSTRACT:

This invention relate to the P
2X
-purinoceptor, its preparation and uses.
The P
2X
-purinoceptor is a ligand-gated ion channel; that is, the receptor itself forms an ion channel which opens when extracellular adenosine 5′-triphosphate (ATP) binds to the receptor. There are five other classes of neurotransmitter receptors (nicotinic acetylcholine, glutamate, glycine, GABA
A
and 5-HT
3
); these form a structurally related superfamily of ligand-gated ion channels (Barnard,
Trends Biochem. Sci.
17, 368-374, (1992)). The P
2X
-receptor now identifies a new family of this type of receptor. The unique structure of this receptor, the widespread distribution of this receptor throughout the body, and the numerous physiological roles this receptor may play, make it an important protein that can be used to identify new, therapeutically effective, compounds for the treatment of a number of pathological states.
In 1929 the eminent physiologist Szent-Gyorgyi described powerful cardiovascular actions of extracellular purine nucleosides (e.g. adenosine) and nucleotides (e.g. ATP) (Drury & Szent-Gyorgyi,
J. Physiol.
68 213-237 (1929)), but it was not until 1972 that pharmacological evidence was provided to suggest the existence of distinct receptors for extracellular ATP (ie. that recognise ATP but not adenosine) (Burnstock,
Pharmacological Reviews
21 509-581 (1972)). The seminal and subsequent work on this area by Burnstock and colleagues was largely unaccepted throughout the 1970s and early 1980s until the development of a range of relatively selective ligands and techniques for directly measuring ATP release overwhelmingly substantiated Burnstock's hypothesis (Barnard et al.,
Trends Pharmacol. Sci.
15 67-70 (1994)). In the past four or five years, unequivocal evidence for the role of ATP as a neurotransmitter has been provided for sympathetic control of blood flow to the intestine and smooth muscle tone (contractility) in genitourinary tissue such as vas deferens, bladder and ureter (Barnard et al. (loc. cit.) and Evans & Surprenant,
Brit. J. Pharmacol.
106 242-249 (1992)). Substantial indirect evidence also exists for the role of ATP as a neurotransmitter in a number of distinct neurones in the spinal cord, autonomic ganglia and certain nuclei in the central nervous system (Bean,
Trends Pharmacol. Sci.
15 67-70 (1992), Evans et al.,
Nature
357, 503-505 (1992) and Edwards et al.,
Nature
359 144-147 (1992)).
Purinoceptors are classified as P
1
(adenosine as ligand) and P
2
(ATP as ligand). The P
2
receptors are subclassified into two broad types—those that are 7-transmembrane receptors that couple to G-proteins (P
2Y
, P
2U
, P
2T
, and perhaps P
2Z
) and those that form a directly gated ion channel (P
2X
). Pharmacological and/or physiological evidence for subtypes of each of these types of receptors exists. The most recent nomenclature for these receptors is shown below.
P
2X
P
2Y
P
2Z
Type
Ligand-gated channel
G-protein coupled
Non-selective pore
Subtype
P
2X
, P
2X2
, P
2X3
P
2Y
, P
2Y2
, P
2Y3
Various P
2
receptors have previously been cloned. P
2Y1
was cloned by the Barnard/Burnstock group (Webb et al.,
FEBS Lett.
324 219-225 (1993)) based on homology with other 7-TM G-protein coupled receptors. This group used PCR technology and primers based on conserved domains of the second and sixth transmembrane regions to screen a mammalian brain cDNA library and, with final success, an embryonic chick whole-brain cDNA library.
P
2Y2
/P
2U
was cloned by the Julius laboratory (Lustig et al.,
Proc. Nat'l. Acad. Sci. USA
90 5113-5117 (1993)) by expression cloning in the oocyte from cDNA obtained from a NG108-15 neuroblastoma cell line.
P
2Y3
/P
2T
was also obtained by the Barnard/Burnstock group using the same probe and embnryonic brain cDNA library used to obtain the P
2Y1
receptor (Barnard et al.,
Trends Pharmacol. Sci.
15 67-70 (1994)).
However, as yet, cloning of the P
2X
receptor has remained an elusive goal. The prior cloning exercises undertaken for the other P
2
receptors do not provide an adequate lead to enable the P
2X
receptor to be cloned. First, all the above purinoceptors are G-protein activation of one or more second messenger systems. There are over 200 currently identified proteins which belong to this 7-TM/G-protein coupled family. Agonists at these receptors activate cascades of intracelluar transduction pathways, often involving several enzymes; the response of the cell is inherently slow (several seconds to minutes) and changes in excitability are subtle if they occur. In contrast, the P
2X
receptor is a fundamentally different type of purinoceptor that incorporates an ion channel. Activation of P
2X
receptors is rapid (milliseconds), has predominately local effects, and brings about immediate depolarisation and excitation.
Secondly, the tissue distribution of the P
2X
receptor is distinctly different from other purinoceptors, and the physiological roles differ from other purinoceptors.
One of the principal established ways to clone a receptor is based on sequence relatedness of the nucleotides that encode the amino acids of the receptor protein; it depends on there being a fairly high level of homology between a known sequence and that of the unknown receptor. This method was used to clone the P
2Y1
from (above). Several laboratories, including that of the applicants, invested significant effort in obtaining the P
2X
receptor using PCR techniques and primers based on conserved regions of various ligand-gated ion channels (ie. nicotinic ACh, GABA, glutamate, 5-HT
3
). This approach failed. With hindsight, this failure can be rationalised, as it can now, but only now, be seen that the structure of the P
2X
receptor bears no homology with any of these ligand-gated ion channels. For the same reason, approaches based on fragment hybridisation would not succeed.
However, by adopting a different approach, it has now been found possible to clone the P
2X
receptor, and it is on this achievement that the present invention is in part based.
According to a principal aspect of the present invention, there is provided a recombinant or isolated DNA molecule encoding a P
2X
receptor, wherein the receptor:
(a) has the amino sequence shown in
FIG. 1
,
FIG. 2
,
FIG. 3
or
FIG. 4
; or
(b) is substantially homologous to the sequence shown in
FIG. 1
,
FIG. 2
,
FIG. 3
or
FIG. 4
;
or a fragment of such a DNA molecule, which fragment includes at least 15 nucleotides taken from nucleotides 1 to 813 shown in
FIG. 1
, the full nucleotide sequences shown in
FIG. 2 and 3
, or from nucleotides 1 to 1744 shown in FIG.
4
.
The sequence shown in
FIG. 1
is a cDNA sequence that encodes a rat vas deferens P
2X
receptor. This sequence is 1837 bases in length and encodes a protein of 399 amino acids. As was determined after the receptor was cloned, approximately one half of the protein-encoding sequence, from nucleotides 814 onwards, had been discovered previously but the function of the previously cloned sequence was not known except that it appeared to be implicated in apoptotic cell death (Owens et al.,
Mol. Cell. Biol.
11 4177-4188 (1991); the Owens et al. sequence lacks a translation initiation site and could not be made into protein. (In
FIG. 1
, the upstream portion of the reported sequence of Owens et al., namely PQLAHGCYPCPPHR, which is not shared with the P
2X
receptor, is shown for comparative purposes and does not form part of the invention.)
Preferably the
FIG. 1
sequence fragments are taken from nucleotides 1-810. Often the
FIG. 4
sequence fragments are taken from nucleotides 1-777.
The sequence shown in
FIG. 2
is a cDNA sequence that encodes a rat superior cervical ganglion P
2X
receptor.
The sequence shown in
FIG. 3
is a cDNA sequence that encodes a rat dorsal root ganglion P
2X
receptor.
The sequence shown in
FIG. 4
is the cDNA sequence that encodes a human P
2X
receptor. The cDNA was isolated from the human urinary bladder using a rat P
2X
probe. It is 2643 bases long and encodes a 399 amino acid protein h

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